Genetic sequences, diagnostic and/or quantification methods and devices for the identification of staphylococci strains

ABSTRACT

The present invention is related to oligonucleotides for the specific identification of Staphylococci species which nucleotide sequence has between 15 and 350 base pairs, preferably between 15 and 45 base pairs, obtained from the “consensus” femA nucleotide sequence (CNS) of FIG. 3 or its complementary strand. The present invention is also related to a method and a diagnostic device using said oligonucleotide for the identification of various types of Staphylococci species strains.

[0001] This patent application is a continuation-in-part (CIP) of U.S.Ser. No. 09/509,234, filed Sep. 25, 2000, the contents of all of whichare incorporated by reference into the present application.

FIELD OF THE INVENTION

[0002] The present invention refers to new genetic sequences, diagnosticand/or quantification methods and devices using said sequences for theidentification of various types of Staphylococci strains as well as thetherapeutical aspects of said genetic sequences.

BACKGROUND OF THE INVENTION

[0003] Increasing incidence of nosocomial infections by multiresistantbacteria (even to antibiotics like vancomycin) is a world-wide concern.Methicillin-resistant coagulase-negative Staphylococci (MR-CNS) and S.aureus (MRSA) express a high level cross-resistance to all β-lactamantibiotics (Ryffel et al. (1990), Refsahl et al. (1992)). They have anadditional low-affinity penicillin-building protein, PBP2a (PBP2′),encoded by the mecA gene. The mecA determinant is found in allmultiresistant staphylococcal species (Chackbart et al. (1989), Suzukiet al. (1992), Vannuffel et al. (1995)) and is highly conserved amongthe different species (Ryffel et al. (1990)).

[0004] Several other chromosomal sites, in which transposon inactivationreduces the level of β-lactam resistance, have been identified in S.aureus (SA) (Hiramatsu (1992), Berger-Bachi et al. (1992), de Lancastreet al. (1994)). The appropriate functioning of these regulator genesrather than the quantity of PBP2a determines the minimal inhibitoryconcentration value and homogeneous expression of resistance ofstaphylococcal isolates (Ryffel et al. (1994), de Lancastre et al.(1994)).

[0005] The femA-femB operon, initially identified in S. aureus, is oneof those genetic factors essential for methicillin resistance(Berger-Bächi et al. (1989)). It is involved in the formation of thecharacteristic pentaglycine side chain of the SA peptidoglycan (Strandenet al. (1997)). Unlike other regulatory genes, femA was shown to retaina strong conservation over time in clinical isolates of MRSA, henceconfirming its key role in cell wall metabolism and methicillinresistance (Hurlimann-Dalel et al. (1992)). In contrast to mecA,femA-femB is present both in the genome of resistant and susceptible SAstrains (Unal et al. (1992), Vannuffel et al. (1995)).

[0006] Often, identification of the Staphylococci is limited to a rapidscreening test for S. aureus, and non-S. aureus isolates are simplyreported as coagulase-negative Staphylococci. In fact, these bacteriaisolates include a variety of species and many different strains(Kleeman et al. (1993)). There is little epidemiological informationrelated to the acquisition and spread of these organisms. This ispotentially due to the lack of an easy and accurate way to identifyspecies and to provide clinically timely informations.

[0007] Several molecular assays designed for detecting femA in SA failedto amplify an homologous sequence in coagulase-negative Staphylococci(Kizaki et al. (1994), Vannuffel et al. (1995)). Nevertheless,low-stringency heterologous hybridisation analysis suggested thepresence of such a structurally related gene in S. epidermidis (SE)(Unal et al. (1992)).

[0008] These data were followed by complete identification and sequenceanalysis of the femA and femB open reading frames in S. epidermidis(Albom et al. (1996)). Intra- and interspecies relatedness of thesegenes and conservation of genomic organisation are therefore consistentwith gene duplication of one of these genes in an ancestral organism andthe possibility of femA phylogenetic conservation in all staphylococcalspecies (Alborn et al. (1996)).

[0009] The complete genetic sequence of the femA gene de S. epidermidis,the protein encoded by the femA gene (FemA) and vectors andmicro-organisms comprising genes encoding the FemA protein are describedin the U.S. Pat. No. 5,587,307.

[0010] Aims of the Invention

[0011] The present invention aims to provide new genetic sequences,methods and devices for the improvement of the identification and/or thequantification of various types of Staphylococci strains through theirfemA-like determinants, which allow by a rapid screening theirepidemiological study.

[0012] Another aim of the invention is to identify similar geneticsequences which may exist in known or not known Staphylococci species orother gram-positive bacterial strains.

[0013] A last aim of the present invention is to provide new sequencesencoding femA proteins of Staphylococci species, their femA proteins,vector(s) comprising said nucleotide sequences and cell (s) transformedby said vector(s) for possible therapeutical applications.

SUMMARY OF THE INVENTION

[0014] The Inventors have identified new DNA and amino acid sequencesfrom new strains of Staphylococcus hominis, Staphylococcus saprophyticusand Staphylococcus haemolyticus. Said new nucleotide sequences allow analignment of these new sequences with the femA gene from Staphylococcipreviously described (S. aureus, S. epidermidis and S. saprophyticus).By the alignment of more than 2 sequences, preferably more than 4sequences, the Inventors have identified for the first time a consensusfemA sequence useful for molecular genotyping of different Staphylococcispecies which was not possible previously, when only few femA sequencesof Staphylococci strains were known.

[0015] Therefore, a first aspect of the present invention is related tothe “consensus” nucleotide sequence as represented in the enclosed FIG.3. With said “consensus” nucleotide sequence, the Inventors were able toprovide oligonucleotides (such as primers or probes) which can be usedfor the genetic amplification, the identification and/or quantificationof various femA sequences which are specific of known or unknownStaphylococci species.

[0016] The femA sequence is known to be involved with the biosynthesisof glycin-containing cross-bridges of the peptidoglycan and thepeptidoglycan organisation is also known to be well conserved amongvarious Staphylococci species and possibly among other gram-positivebacteria.

[0017] Therefore, it is also possible to use the new “consensus” femAsequence and said new oligonucleotides extrapolated from the alignmentof the sequences presented in FIG. 3, for the molecular genotyping ofother Staphylococci species and possibly other gram-positive bacteria.It is also known that the femA sequence is similar to the femB sequence.Therefore, these oligonucleotides could also be used for the moleculargenotyping of femB genes of different Staphylococci species or othergram-positive bacteria.

[0018] Another aspect of the present invention concerns the possibletherapeutical uses of new femA nucleotide sequences isolated from thestrains S. hominis, S. saprophyticus, S. haemolyticus, S. lugdunensis,S. xylosus, S. capitis, S. schleiferi and S. sciuri having a nucleotideor amino acid sequence which presents more than 85%, preferably morethan 90% homology or 100% homology with the genetic sequences presentedin the FIGS. 6 to 13, their complementary strand and functional variantsthereof. Functional variants of said amino acid sequences are peptideswhich contain one or more modifications to the primary amino acidssequence and retain the activity of the complete and wild type femAmolecule. Variants of the peptide are obtained by nucleotidic sequenceswhich differ from the above-identified described sequences by adegeneration of their genetic code or are sequences which hybridise withsaid sequences or their complementary strand, preferably under stringentconditions such as the ones described in the document Sambrook et al.,§§ 9.47-9.51 in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0019] A further aspect of the present invention concerns therecombinant vector (i.e. constructions into which the sequence of theinvention may be inserted for transport in different geneticenvironments and for expression in a host cell, such as a phagemide, avirus, a plasmid, a cationic vesicle, a liposome, etc.) comprising saidnucleotide sequences and their complementary strands, or thecorresponding RNA sequences, possibly linked to one or more regulatorysequences or markers (resistance to antibiotics, enzyme codingsequences, . . . ) active into a cell.

[0020] Similarly, the nucleic acid sequence according to the inventionmay be obtained by synthetic methodology well known by the personskilled in the art, such as the one described by Brown et al. (“Methodof Enzymology”, Acad. Press, New-York, No. 68 pp. 109-151 (1979)) or byconventional DNA synthesising apparatus such as the applied biosystemmodel 380A or 380B DNA synthesiser.

[0021] Other aspects of the present invention concern the recombinanthost (prokaryotic) cell transformed by said vector and the purified(possibly recombinant) proteins or peptides encoded by said nucleic acidsequences, possibly linked to a carrier molecule such as BSA andobtained by said cells. Said recombinant proteins or peptides could beobtained by genetic engineering or could be obtained by synthesis (seeU.S. Pat. No. 5,587,307 incorporated herein by reference) and maycomprise residues enhancing their stability (resistance to hydrolysis byproteases, etc.) such as the one described by Nachman et al. (Regul.Pept. Vol. 57, pp. 359-370 (1995)).

[0022] A preferred vector for expression in a E. coli host cell isderived from the E. coli plasmid pET-11A available from Novagen Inc.(Catalogue No. 69436-A). The transformation technique used with theabove-identified vector has been described in the U.S. Pat. No.5,587,307.

[0023] A further aspect of the present invention concerns the inhibitor(used to possibly treat (with addition of antibiotics) antibioticsresistance bacteria) directed against said proteins, peptides or nucleicacid molecules. Advantageously, said inhibitor is a antibody, preferablya monoclonal antibody, or an antisense nucleotide molecule, such as aribozyme, which could be present in a vector in order to block theexpression of said femA nucleotide sequences.

[0024] A last aspect of the present invention concerns thepharmaceutical composition, preferably a vaccine, against Staphylococciinfections in an animal, including a human, comprising apharmaceutically acceptable carrier and a sufficient amount of an activecompound selected from the group consisting of said nucleic acidmolecules, vectors, recombinant host cells transformed by saidvector(s), inhibitors (directed against said proteins, peptides ornucleic acid molecules) and a mixture thereof.

[0025] Another aspect of the present invention concerns oligonucleotideswhich are (DNA) sequences having between 15 and 350 base pairs,preferably between 17 and 250 base pairs (such as primers or probes)obtained from the consensus sequence of FIG. 3 or its complementarystrand. Preferably, said oligonucleotides are primers having between 15and 45 base pairs, more preferably between 17 and 25 base pairs.

[0026] According to a first embodiment of the present invention, saidoligonucleotide is a primer having between 15 and 45 base pairs, whichpresents more than 60%, advantageously more than 70%, preferably morethan 80%, more specifically more than 90% homology with (fragments of)the “consensus” femA nucleotide sequence (CNS) identified in the FIG. 3.

[0027] Therefore, the oligonucleotides according to the invention arenew sequences or preferred fragments of known sequences of S. aureus, S.epidermidis or S. simulans but not the complete wild type known femAnucleotide sequence.

[0028] Preferably, the oligonucleotide according to the invention isselected from the group consisting of the following nucleotidesequences:

[0029] ANAATGAANTTTACNAATTTNACNGCNANAGANTT (SEQ ID NO: 2) and moreparticularly femS1 TAATGAAGTTTACAAAATTT (SEQ ID NO: 3) or femS2TAATGAAGTTTACNAAATTT (SEQ ID NO: 4)

[0030] ATGNCNNANAGNCATTTNACNCANA (SEQ ID NO: 5) and more particularlyfemU1 (“universal” sequence sense of the multiplex PCR):TGCCATATAGTCATTTACGC (SEQ ID NO: 6)

[0031] TAGTNGGNATNAANAANAANNATAANGANGTNATTGC (SEQ ID NO: 7)

[0032] GTNCCNGTNATGAAANTNTTNAANTANTTTTATTC (SEQ ID NO: 8)

[0033] AATGCNGGNNANGATTGG (SEQ ID NO: 9)

[0034] GNAANNGNAANACNAAAAAAGTNNANAANAATGGNGTNAAAGT (SEQ ID NO: 10) andmore particularly fsq1S (et 1AS): AAAAAGTTCAAAAAATGG (SEQ ID NO: 11) andfsq2S (and 2AS): AAAAAGTACAAAAAATGG (SEQ ID NO: 12)

[0035] AAGANGANNTNCCNATNTTNNGNTCATTNATGGANGATAC (SEQ ID NO: 13)

[0036] TATATNNANTTTGATGANTA (SEQ ID NO: 14)

[0037] AANGANATNGANAAANGNCCNGANAANAAAAA (SEQ ID NO: 15) and moreparticularly fsq3S (and 3AS): AAAGATATTGAAAAACGA (SEQ ID NO: 16), fsq4S(and 4AS): AAAGATATTGAAAAGAGACC (SEQ ID NO: 17), fsq5S (and 5AS):AAAGATATCGAGAAAGAC (SEQ ID NO: 18) and fsq6S (and 6AS)AAAGACATCGACAAGCGT (SEQ ID NO: 19).

[0038] ANCATGGNAANGAATTACCNAT (SEQ ID NO: 20) and more particularly fem1(primer for the production of a probe and of marked amplicons forreverse hybridisation experiment): GAACATGGTAATGAATTAC (SEQ ID NO: 21)

[0039] AATCCNTNTGAAGTNGTNTANTANGCNGGTGG (SEQ ID NO: 22)

[0040] AGNTATGCNNTNCAATGGNNNATGATTAANTATGC (SEQ ID NO: 23)

[0041] TTTANNGANGANGCNGAAGATGNNGGNGTNNTNAANTTNAAAAA (SEQ ID NO: 24) andmore particularly fem3bio (primer for the production of a probe and ofmarked amplicons for reverse hybridization experiment):TTTACTGAAGATGCTGAAGA (SEQ ID NO: 25)

[0042] GTTGGNGANTTNNTNAAACC (SEQ ID NO: 26) and more particularly fem2(primer for the production of a probe and of marked amplicons forreverse hybridisation experiment): GTTGGTGACTTTATTAAACC (SEQ ID NO: 27)

[0043] ATGAAATTTACAGAGTTAA (=femAS1) (SEQ ID NO: 28).

[0044] In addition, the following universal primer may also be used inthe present invention: GCCATACAGTCATTTCACGC (=uni) (SEQ ID NO: 65).

[0045] Said primer(s) will be designated hereafter as “universalprimer(s)”.

[0046] A further aspect of the present invention concerns theoligonucleotide being either a primer or a probe as above-described,having between 15 and 350 base pairs, preferably between 17 and 250 basepairs, or a primer having between 15 and 45 base pairs, more preferablybetween 17 and 25 base pairs, which will be designated hereafter as“specific primer(s)”, having a nucleotide sequence which presents lessthan 50%, advantageously less than 40%, preferably less than 30%, morespecifically less than 20% homology with (fragments of) the “consensus”femA nucleotide sequence (CNS) identified in the FIG. 3 and with anotherfemA nucleotide sequence specific for other Staphylococci strains.

[0047] Advantageously, said “specific primer” is selected from the groupconsisting of the following nucleotide sequences: ACAGCAGATGACATCATT(SEQ ID NO: 29) TAATGAAAGAAATGTGCTTA (SEQ ID NO: 30) ACACAACTTCAATTAGAAC(SEQ ID NO: 31) AGTATTAGCAAATGCGG (SEQ ID NO: 32) ATGCATATTTTCCGTAA (SEQID NO: 33) CAGCAGATGACATCATT (SEQ ID NO: 34) CATCTAAAGATATATTAAATGGA(SEQ ID NO: 35) AGTATTAGCAAATGCGGGTCAC (SEQ ID NO: 36)CAACACAACTTCAATTAGAA (SEQ ID NO: 37)

[0048] In addition, the following specific femA primers can be used in amultiplex protocol for staphylococcal differential diagnosis:CAGCAGATGACATCATTA (SEQ ID NO: 66) for identification of S. aureus,AACCGAAACGAAAGAATT (SEQ ID NO: 67) for identification of S.haemolyticus, and CAACACAACTTCAATTAGAAC (SEQ ID NO: 68) foridentification of S. hominis.

[0049] Further, the following primers or probes can be used for each ofthe following staphylococcal species, in the method or kit according tothe present invention for the identification and/or quantification of aStaphylococci species, which may present resistance to antibiotics andwhich is present in a sample: S. aureus sbs. anaerobius:ATTTAAAATATCACGCTCTTCGTTTAG, (SEQ ID NO:69) S. epidermidis:ATTAAGCACATTTCTTTCATTATTTAG, (SEQ ID NO:70) S. haemolyticus:ATTTAAAGTTTCACGTTCATTTTGTAA, (SEQ ID NO:71) S. hominis:ATTTAATGTCTGACGTTCTGCATGAAG, (SEQ ID NO:72) S. saprophyticus:ACTTAATACTTCGCGTTCAGCCTTTAA, (SEQ ID NO:73) S. capitis:ATTAAGAACATCTCTTTCATTATTAAG, (SEQ ID NO:74) S. cohnii sbs. urealyticum:ACTTAACACTTCACGCTCTGACTTGAG, (SEQ ID NO:75) S. gallinarum:ACTTAAAACTTCACGTTCAGCAGTAAG, (SEQ ID NO:76) S. intermedius:GTGGAAATCTTGCTCTTCAGATTTCAG, (SEQ ID NO:77) S. lugdunensis:TTCTAAAGTTTGTCGTTCATTCGTTAG, (SEQ ID NO:78) S. schielferi:TTTAAAGTCTTGCGCTTCAGTGTTGAG, (SEQ ID NO:79) S. sciuri:GTTGTATTGTTCATGTTCTTTTTCTAA, (SEQ ID NO:80) S. simulans:TTCTAAATTCTTTTGTTCAGCGTTCAA, (SEQ ID NO:81) S. warneri:AGTTAAGGTTTCTTTTTCATTATTGAG, (SEQ ID NO:82) S. xylosus:GCTTAACACCTCACGTTGAGCTTGCAA. (SEQ ID NO:83)

[0050] The oligonucleotides according to the invention are selectedaccording to their physiochemical properties in order to avoidcross-hybridisation between themselves. Said primers are notcomplementary to each other and they contain a similar percentage ofbases GC.

[0051] Said oligonucleotides are used in an identification and/orquantification method of one or more Staphylococcus species and possiblyother gram-positive bacteria. In a preferred embodiment, saidoligonucleotides are used in an identification and/or quantificationmethod of at least two different Staphylococcus species and possiblyother gram-positive bacteria.

[0052] Therefore, another aspect of the present invention is related toan identification and/or quantification method of a Staphylococcispecies which may present resistance to one or more antibiotic(s), andis possibly combined with a method for the identification of aresistance to antibiotics, especially β-lactam antibiotics, (forinstance through the identification of a variant of the mecA gene asdescribed by Vannuffel et al. (1998)).

[0053] In another embodiment, the present invention is related to anidentification and/or quantification method of at least two differentStaphylococci species. The diagnostic device for the identification ofat least two Staphylococci species comprises preferably at least oneoligonucleotide which has more than 60% homology to SEQ ID NO:1 and atleast one isolated or purified oligonucleotides which has less than 50%homology to SEQ ID NO:1.

[0054] The method for the detection, the identification and/or thequantification of a bacteria, preferably a staphylococcal species,comprises the steps of:

[0055] obtaining a nucleotide sequence from said bacteria present in asample, preferably a biological body sample obtained from a patient suchas blood, serum, dialyse liquid or cerebrospinal liquid, or from anyother bacteriological growth medium,

[0056] possibly purifying said nucleotide sequence from possiblecontaminants,

[0057] possibly amplifying by known genetic amplification techniquessaid nucleotide sequence with one or more universal oligonucleotide(s)(universal primer(s)) according to the invention, and

[0058] identifying the specific gram-positive bacteria species,preferably the specific Staphylocossi species:

[0059] by a comparative measure of the length of the (possiblyamplified) nucleotide sequence or

[0060] by reverse hybridisation of the (possibly amplified) nucleotidesequence with one or more specific oligonucleotide(s) (specific probe(s)or primer(s)) according to the invention which are specific of saidbacteria, said oligonucleotide(s) being preferably immobilised on asolid support.

[0061] The comparative measure of the length of a possibly amplifiednucleotide sequences can be performed by the analysis of their migration(compared with a known ladder) upon an electrophoresis gel.

[0062] Preferably, the genetic amplification technique is selected fromthe group consisting of PCR (U.S. Pat. No. 4,965,188), LCR (Landgren etal., Sciences, 241, pp. 1077-1080 (1988)), NASBA (Kievits et al., J.Virol. Methods, 35, pp. 273-286 (1991)), CPR (patent WO95/14106) or ICR.

[0063] The specific detection of the possibly amplified nucleotidesequences can be obtained by the person skilled in the art by usingknown specific gel electrophoresis techniques, in situ hybridisation,hybridisation on solid support, in solution, on dot blot, by Northernblot or Southern blot hybridisation, etc.

[0064] Advantageously, the probes which are specific of the bacteria areimmobilised on a solid support according to the method described in theinternational patent application WO98/11253 incorporated herein byreference.

[0065] Said specific oligonucleotides (probes or “elongated” primers)have a length comprised between 50 and 350 base pairs, preferablybetween 120 and 250 base pairs, and are fixed to the solid support by aterminal 5′ phosphate upon an amine function of the solid support bycarbodiimide reaction (as described in the document WO98/11253incorporated herein by reference).

[0066] The solid support can be selected from the group consisting ofcellulose or nylon filters, plastic supports such as 96-well microtiterplates, microbeads, preferably magnetic microbeads, or any other supportsuitable for the fixation of a nucleotide sequence.

[0067] The method according to the invention can be advantageouslycombined with another specific detection step of a possible resistanceto antibiotics, especially β-lactam antibiotics (for instance throughthe identification by the above-described technique of variants of themecA gene as described by Vannuffel et al. (1998)).

[0068] The present invention concerns also a diagnostic and/orquantification device or kit for the identification and/or thequantification of a Staphylococcus species or other gram-positivebacteria, comprising the oligonucleotides according to the invention andpossibly all the media necessary for the identification of a (possiblyamplified) nucleotide sequence of said bacteria through any one of theabove-described methods. In another embodiment, the present inventionconcerns also a diagnostic and/or quantification device or kit for theidentification and/or the quantification of at least two differentStaphylococcus species or other gram-positive bacteria, comprising theoligonucleotides according to the invention and possibly all the medianecessary for the identification of a (possibly amplified) nucleotidesequence of said bacteria through any one of the above-describedmethods.

[0069] Advantageously, the method and device according to the inventionare adapted for the quantification of said Staphylococci strains by theuse of a “internal or external standard sequence”, preferably the onedescribed in the patent application WO98/11253 incorporated herein byreference.

[0070] Therefore, according to a first embodiment of the presentinvention, the nucleic acid sequence from a Staphylococcus species, forinstance Staphylococcus aureus, is amplified by a “universal primer” andby a “specific primer” which is specific for S. aureus. Theidentification of S. aureus will be obtained upon an agaroseelectrophoresis gel wherein the amplified nucleotide sequence (shorterthan the amplified nucleotide sequence of another Staphylococci speciessuch as S. epidermidis) and identified by the use of a comparativeladder.

[0071] According to another embodiment of the present invention, aStaphylococcus species (such as S. aureus) is identified by reversehybridisation of the amplified nucleotide sequence with a probe which isspecific of said bacteria and which is immobilised on a solid supportsuch as filter.

[0072] The present invention will be described in details in thefollowing non-limiting examples, in reference to the Figures describedhereafter.

SHORT DESCRIPTION OF THE DRAWINGS

[0073] The FIG. 1 represents 5 partially overlapping fragments of thefemA genes from S. hominis, S. saprophyticus and S. haemolyticusobtained by PCR amplification.

[0074] The FIGS. 2a & b represent the alignment of the nucleotidesequences of femA genes from S. hominis, S. saprophyticus, S. aureus, S.epidermidis and S. haemolyticus.

[0075] The FIG. 3 represents the consensus sequence according to theinvention.

[0076] The FIG. 4 represents the result of differential diagnosisbetween different strains of Staphylococci by reverse hybridisation.

[0077] The FIG. 5 represents amplification of CNS species underuniversal conditions.

[0078] The FIGS. 6a & b represent the complete femA wild type geneticsequence of the strain S. haemolyticus.

[0079] The FIGS. 7a & b represent the complete femA wild type geneticsequence of the strain S. lugdunensis.

[0080] The FIGS. 8a & b represent the complete femA wild type geneticsequence of the strain S. xylosus.

[0081] The FIGS. 9a & b represent the complete femA wild type geneticsequence of the strain S. capitas.

[0082] The FIG. 10a & b represent the complete femA wild type geneticsequence of the strain S. schleiferi.

[0083] The FIGS. 11a & b represent the complete femA wild type geneticsequence of the strain S. sciuri.

[0084] The FIG. 12 represents the complete femA wild type geneticsequence of the strain S. hominis.

[0085] The FIG. 13 represents the complete femA wild type geneticsequence of the strain S. saprophyticus.

EXAMPLES Example 1 Sequencing Strategy

[0086] Fragments of the femA genes from S. hominis and S. saprophyticushave been obtained by PCR amplification, in low stringency annealingconditions. Primers used for amplification are matching the potentiallyconserved regions and have been designed according to sequenceshomologies between S. aureus, S. sapropyticus and S. epidermidis femAnucleotide sequences. For both S. hominis and S. saprophyticus species,5 partially overlapping fragments have been synthesised allowing thesequencing of the entire femA genes (FIG. 1).

Example 2 Identification of a Consensus Sequence

[0087] Alignment of the nucleotide sequences of femA genes from S.hominis and S. saprophyticus as well as with femA genes sequenced todate from S. aureus (GenBank accession number M23918), S. epidermidis(GenBank accession number U23713) and S. haemolyticus is presented inFIG. 3 and has allowed to propose a “consensus” femA nucleotide sequence(CNS) whose genomic organisation displays highly conserved regionsflanked by variable ones. On this basis, interspecies phylogeneticvariations could be exploited to design genotyping strategies forspecies-specific identification of Staphylococci. The “consensus”sequence is therefore a powerful molecular tool for specific diagnosticof staphylococcal infections.

Example 3 Sequencing of other Staphylococcal femA Genes

[0088] The consensus sequence can be exploited for designing universalprimers allowing the production, under permissive annealing conditions,of overlapping PCR products whose sequencing will identify the entirefemA sequence.

Example 4 Differential Diagnosis between S. aureus. S. epidermidis, S.hominis and S. saprophyticus by Reverse Hybridisation

[0089] The Inventors have set up a reverse hybridisation assay for rapidand combined identification of the most clinically relevantStaphylococci species, and their mecA status. Two sets of primers,chosen in a conserved domain of the consensus sequence (bioU1-bioU3 andfem1-fem3bio), amplifying a 286 and bio-220 bp fragments, respectively)were synthesised. Species-specificity of femA amplicons was insured bythe genomic variability between the conserved regions. FemA probes wereimmobilised on nylon strips. Hybridisation was performed withbiotinylated femA PCR fragments from the strain of interest. Thestrategy was first assessed with ATCC strains (S. aureus, S.epidermidis, S. hominis and S. saprophyticus) (FIG. 4). Specificity wasidentified by standard methods. Accuracy was 100% for speciesidentification.

Example 5 Differential Diagnosis between Staphylococcal Species

[0090] This assay is able to identify any staphylococcal species iffollowing requirements are fulfilled:

[0091] primers fem1, fem2 and fem3bio are universal for Staphylococci;

[0092] there is a wide enough phylogenetic variation between any CNSspecies to promote a specific hybridisation.

[0093] The first requirement is fulfilled for, i.e., S. haemolyticus, S.capitis, S. cohnii, S. xylosus, S. simulans, S. lugdunensis, S.schleiferi and S. warneri strains (FIG. 5).

Example 6 Multiplex Amplification of femA and mecA Genetic Determinantsfor a Molecular Diagnosis of a Specific Staphylococcal Infection

[0094] A total of 48 patients treated in 4 contiguous intensive caresunits were included in the study. Endotracheal aspirates (ETA) werecollected from the patients and submitted to the multiplex PCR analysisaccording to the technique described by Vannuffel et al. (1995).Clinical specimens were homogenised in 5 ml of TE buffer (20 mM TRISHCl, pH 8.0, 10 mM EDTA) containing 2% (w/v) SDS.

[0095] The homogenate (1.5 ml) was then centrifuged for 5 minutes at7500×g. The cellular pellet was washed once with TE buffer lysed in thepresence of 1% (v/v) Triton X-100 and 50 μg of lysostaphin (Sigma) andincubated for 15 minutes at 37° C. Lysis was completed by adding 100 μgof proteinase K (Boehringer). The lysate was incubated for another 5minutes at 55 ° C. and 5 minutes at 95° C., and centrifuged at 4000×gfor 5 minutes.

[0096] In order to purify bacterial DNA, 200 μl of supernatant were thenfiltered on a Macherey-Nagel Nucleospin C+T® column and eluted with 200μl sterile H2O. Two different amounts of DNA suspension (2 μl and 200μl) were submitted to multiplex PCR amplification with the primers5′-TGGCTATCGTGTCACAATCG-3′ (SEQ ID NO: 38)and 5′-CTGGAACTTGTTGAGCAGAG-3′(SEQ ID NO: 39) for mecA and the above-described primers for femA,yielding different fragments.

[0097] femA and mecA signals were found in specimens containing eithersusceptible S. aureus (n=10) and methycillin-resistantcoagulase-negative Staphylococci (n=6) respectively. On the other hand,no signal was obtained from ETA gram-negative bacteria (n=5) as well asMS-CNS (n=6) and from 5 ETA containing normal pharyngeal flora.

[0098] This multiplex PCR strategy for detecting Staphylococci in ETAwas completed in less than 6 hours either on the day of the samples'collection. This is an advantage with respect to the time required toconventional identification and susceptibility tests (48 to 72 hours).

Example 7 Amplification, Cloning and Sequencing of other femA Genes

[0099] Two primers were selected among the conserved parts of theconsensus sequence for the amplification of the femA gene.

[0100] These primers are femS1, femS2 and femAS1 (anti-sense primer).ADN from strains of Staphylococcus hominis, saprophyticus, haemolyticus,lugdunensis, schleiferi, sciuri, xylosus, simulans, capitis, gallinarum,cohnii and wameri were amplified from said primers and amplificationfragments were cloned in the vector pCR®-XLTOPO and introduced byelectroporation in E. coli cells TOP10 (TOPO XL PCR Cloning Kit®,Invitrogen, Carlsbad, Calif.).

[0101] Amplified fragments of strain S. lugdunensis, schleiferi, sciuri,xylosus, and capitis were sequenced by Taq Dye Deoxy Terminator Cycle®sequencing on a ABI 277 DNA sequencer® (PE Applied Biosystems, FosterCity, Calif.) by the following primers:

[0102] femS1 or femS2 or femAS1

[0103] fsq1S and fsq1AS

[0104] fsq2S and fsq2AS

[0105] fsq3S and fsq3AS

[0106] fsq4S and fsq4AS

[0107] fsq5S and fsq5AS

[0108] fsq6S and fsq6AS

References

[0109] 1. Alborn W. E. Jr et al., Gene 180: 177-81 (1996)

[0110] 2. Berger-Bächi B. et al, Mol Gen Genet 219: 263-9 (1989)

[0111] 3. Berger-Bächi B. et al., Antimicrob. Agents Chemother. 36:1367-73 (1992)

[0112] 4. Chackbart et al., Antimicrobial Agent Chemotherapy 33: 991-999(1989)

[0113] 5. de Lancastre H. et al., Antimicrob. Agents Chemother. 38:2590-8 (1994)

[0114] 6. Hiramatsu K. et al., FEBS Letters 298: 133-6 (1992)

[0115] 7. Hurlimann-Dalel R. L. et al., Antimicrob. Agents Chemother.36: 6+17-21 (1992)

[0116] 8. Kizaki M. et al., J. Hosp. Infect. 28: 287-95 (1994)

[0117] 9. Kleeman K. T. et al., J. Clin. Microbiol. 31: 1318-1321 (1993)

[0118] 10. Refshal K. et al., J. Hosp. Infect. 22(1): 19-31 (1992)

[0119] 11. Ryffel C. et al., Gene 94: 137-8 (1990)

[0120] 12. Ryffel C. et al., Antimicrob. Agents Chemother. 38: 724-8(1994)

[0121] 13. Rupp M. E. et al., Clin. Infectious Diseases 19: 231-245(1994)

[0122] 14. Stranden A. L. et al., J. Bacteriol. 179: 9-16 (1997)

[0123] 15. Suzuki E. et al., Antimicrob. Agents Chemother. 36: 429-34(1992)

[0124] 16. Unal S. et al., J. Clin. Microb. 30: 1685-1691 (1992)

[0125] 17. Vannuffel P. et al., J. Clin. Microb. 33: 2864-2867 (1995)

[0126] 18. Vannuffel . et al., J. Clin. Microb. 36: 2366-2368 (1998)

1 83 1 1328 DNA Staphylococcus femA Consensus Sequence misc_feature(1)...(1328) n=any nucleotide 1 nnnnnnnnnn nnnanaatga antttacnaatttnacngcn anaganttnn gnnnntntac 60 ngannnnatg ncnnanagnc atttnacncanannnnngnn nantangann tnaannttgc 120 nnannnnnnn ganncncann tagtnggnatnaanaanaan nataangang tnattgcngc 180 ntgnntnntn acngcngtnc cngtnatgaaantnttnaan tanttttatt cnaanngngg 240 nccngtnatn gattntnana annnaganctngtncantnn ttctttaang anttnnnnaa 300 ntatntnaaa nannannntn nnntatanntnnnnntngan ccntanntnn cntatcaata 360 nnnnaatcat ganggngann tnnnngnnaatgcnggnnan gattggntnt tngatnannt 420 nnnnnnnntn ggntntnanc annnnggnttnnnnannggn tttganccnn tnnnncaaat 480 nngntnncan tcngtnntan atttannnnnnaaaannncn nanganntnn tnaannnnat 540 ggatngnntn ngnaanngna anacnaaaaaagtnnanaan aatggngtna aagtnnnntt 600 nntnnnnnaa ganganntnc cnatnttnngntcattnatg gangatacnn cnganncnaa 660 ngnnttnnnn gatngngang annnnttntantanaanngn tnnnnnnatt nnaaagannn 720 ngtnntngtn ccnntngcnt atatnnantttgatgantan ntnnnngaan tnnannnnga 780 nngnnannnn ntnantaaag annnnaanaaagcnntnaan ganatngana aangnccnga 840 naanaaaaan gcnnnnaana annnnnnnaanntnnaanan caantnnnng cnaannanca 900 aaanntnnan gangnnannn nnntnnaannnnancatggn aangaattac cnatntcngc 960 ngnntncttn ntnatnaatc cntntgaagtngtntantan gcnggtggna cntcnaatnn 1020 ntnnngncan ttngcnggna gntatgcnntncaatggnnn atgattaant atgcnntnna 1080 ncatnnnatn nanngntana atttntatggnnttagnggt nantttanng angangcnga 1140 agatgnnggn gtnntnaant tnaaaaanggntnnnatgcn ganntnntng antangttgg 1200 nganttnntn aaaccnatna anaanccnntntannnnnnn tatannncan tnaaaaannt 1260 nnannnnann nnnnnntann nannnnnnnannnnannnnn nnnnnnatga aatttacaga 1320 gttaannn 1328 2 35 DNA ArtificialSequence Primer 2 anaatgaant ttacnaattt nacngcnana gantt 35 3 20 DNAArtificial Sequence Primer 3 taatgaagtt tacaaaattt 20 4 20 DNAArtificial Sequence Primer 4 taatgaagtt tacnaaattt 20 5 25 DNAArtificial Sequence Primer 5 atgncnnana gncatttnac ncana 25 6 20 DNAArtificial Sequence Primer 6 tgccatatag tcatttacgc 20 7 37 DNAArtificial Sequence Primer 7 tagtnggnat naanaanaan nataangang tnattgc 378 35 DNA Artificial Sequence Primer 8 gtnccngtna tgaaantntt naantanttttattc 35 9 18 DNA Artificial Sequence Primer 9 aatgcnggnn angattgg 18 1043 DNA Artificial Sequence Primer 10 gnaanngnaa nacnaaaaaa gtnnanaanaatggngtnaa agt 43 11 18 DNA Artificial Sequence Primer 11 aaaaagttcaaaaaatgg 18 12 18 DNA Artificial Sequence Primer 12 aaaaagtaca aaaaatgg18 13 40 DNA Artificial Sequence Primer 13 aagangannt nccnatnttnngntcattna tggangatac 40 14 20 DNA Artificial Sequence Primer 14tatatnnant ttgatganta 20 15 32 DNA Artificial Sequence Primer 15aanganatng anaaangncc nganaanaaa aa 32 16 18 DNA Artificial SequencePrimer 16 aaagatattg aaaaacga 18 17 20 DNA Artificial Sequence Primer 17aaagatattg aaaagagacc 20 18 18 DNA Artificial Sequence Primer 18aaagatatcg agaaagac 18 19 18 DNA Artificial Sequence Primer 19aaagacatcg acaagcgt 18 20 22 DNA Artificial Sequence Primer 20ancatggnaa ngaattaccn at 22 21 19 DNA Artificial Sequence Primer 21gaacatggta atgaattac 19 22 32 DNA Artificial Sequence Primer 22aatccntntg aagtngtnta ntangcnggt gg 32 23 35 DNA Artificial SequencePrimer 23 agntatgcnn tncaatggnn natgattaan tatgc 35 24 44 DNA ArtificialSequence Primer 24 tttanngang angcngaaga tgnnggngtn ntnaanttna aaaa 4425 20 DNA Artificial Sequence Primer 25 tttactgaag atgctgaaga 20 26 20DNA Artificial Sequence Primer 26 gttggngant tnntnaaacc 20 27 20 DNAArtificial Sequence Primer 27 gttggtgact ttattaaacc 20 28 19 DNAArtificial Sequence Primer 28 atgaaattta cagagttaa 19 29 18 DNAArtificial Sequence Primer 29 acagcagatg acatcatt 18 30 20 DNAArtificial Sequence Primer 30 taatgaaaga aatgtgctta 20 31 19 DNAArtificial Sequence Primer 31 acacaacttc aattagaac 19 32 17 DNAArtificial Sequence Primer 32 agtattagca aatgcgg 17 33 17 DNA ArtificialSequence Primer 33 atgcatattt tccgtaa 17 34 17 DNA Artificial SequencePrimer 34 cagcagatga catcatt 17 35 23 DNA Artificial Sequence Primer 35catctaaaga tatattaaat gga 23 36 22 DNA Artificial Sequence Primer 36agtattagca aatgcgggtc ac 22 37 20 DNA Artificial Sequence Primer 37caacacaact tcaattagaa 20 38 20 DNA Artificial Sequence Primer 38tggctatcgt gtcacaatcg 20 39 20 DNA Artificial Sequence Primer 39ctggaacttg ttgagcagag 20 40 1305 DNA Staphylococcus haemolyticus femACDS (4)...(1266) 40 ata atg aag ttt aca aat tta aca gct aca gag ttt ggcaat tat aca 48 Met Lys Phe Thr Asn Leu Thr Ala Thr Glu Phe Gly Asn TyrThr 1 5 10 15 gat aag atg cca tat agt cat ttc aca caa atg act gaa aactat gag 96 Asp Lys Met Pro Tyr Ser His Phe Thr Gln Met Thr Glu Asn TyrGlu 20 25 30 atg aaa gtt gca aat aaa aca gaa act cac tta gtt ggt ata aaaaat 144 Met Lys Val Ala Asn Lys Thr Glu Thr His Leu Val Gly Ile Lys Asn35 40 45 aaa gat aat gag gtt att gca gcc tgc atg ttg aca gca gta cca gtc192 Lys Asp Asn Glu Val Ile Ala Ala Cys Met Leu Thr Ala Val Pro Val 5055 60 atg aaa ttt ttt aag tac ttt tat tct aac cga gga cct gta att gat240 Met Lys Phe Phe Lys Tyr Phe Tyr Ser Asn Arg Gly Pro Val Ile Asp 6570 75 tat gat aat aga gag ctt gtt cac ttt ttc ttt aat gag tta aca aag288 Tyr Asp Asn Arg Glu Leu Val His Phe Phe Phe Asn Glu Leu Thr Lys 8085 90 95 tat tta aaa cag cat aat tgt cta tat gtt cga gtt gac cct tat tta336 Tyr Leu Lys Gln His Asn Cys Leu Tyr Val Arg Val Asp Pro Tyr Leu 100105 110 cca tat caa tat tta aat cat gat ggt gaa att aca ggt aat gct ggt384 Pro Tyr Gln Tyr Leu Asn His Asp Gly Glu Ile Thr Gly Asn Ala Gly 115120 125 aat gat tgg ttc ttt gat aag atg aag cat ctc gga ttt gaa cat gaa432 Asn Asp Trp Phe Phe Asp Lys Met Lys His Leu Gly Phe Glu His Glu 130135 140 ggc ttt act aaa ggt ttt gat ccg att aaa caa atc cga tat cat tct480 Gly Phe Thr Lys Gly Phe Asp Pro Ile Lys Gln Ile Arg Tyr His Ser 145150 155 gtt tta gat tta aaa aat aaa aca tct aaa gat ata tta aat gga atg528 Val Leu Asp Leu Lys Asn Lys Thr Ser Lys Asp Ile Leu Asn Gly Met 160165 170 175 gat agt cta cgt aaa cgt aat act aaa aaa gtt caa aaa aat ggtgtg 576 Asp Ser Leu Arg Lys Arg Asn Thr Lys Lys Val Gln Lys Asn Gly Val180 185 190 aaa gtt aag ttc tta tca gaa gaa gaa ctt cca atc ttc cgt tcattt 624 Lys Val Lys Phe Leu Ser Glu Glu Glu Leu Pro Ile Phe Arg Ser Phe195 200 205 atg gaa gat aca acc gaa acg aaa gaa ttc caa gat aga gat gatagt 672 Met Glu Asp Thr Thr Glu Thr Lys Glu Phe Gln Asp Arg Asp Asp Ser210 215 220 ttc tat tat aat cgc tat aga cat ttc aaa gat cac gtg ctt gtacca 720 Phe Tyr Tyr Asn Arg Tyr Arg His Phe Lys Asp His Val Leu Val Pro225 230 235 cta gct tat att aag ttt gat gag tac atc gaa gaa tta caa aatgaa 768 Leu Ala Tyr Ile Lys Phe Asp Glu Tyr Ile Glu Glu Leu Gln Asn Glu240 245 250 255 cgt gaa act tta aat aaa gat gtt aat aaa gct tta aaa gatatt gaa 816 Arg Glu Thr Leu Asn Lys Asp Val Asn Lys Ala Leu Lys Asp IleGlu 260 265 270 aaa cga cca gac aat aaa aag gca ttt aat aaa aaa gaa aatctt gaa 864 Lys Arg Pro Asp Asn Lys Lys Ala Phe Asn Lys Lys Glu Asn LeuGlu 275 280 285 aaa caa tta gat gcc aat caa caa aaa tta gac gag gct aaaaaa tta 912 Lys Gln Leu Asp Ala Asn Gln Gln Lys Leu Asp Glu Ala Lys LysLeu 290 295 300 caa gcc gaa cat ggt aat gaa tta cca att tca gca ggt ttcttc ttt 960 Gln Ala Glu His Gly Asn Glu Leu Pro Ile Ser Ala Gly Phe PhePhe 305 310 315 att aat cca ttt gaa gtt gtt tat tat gca ggt gga act tctaat aaa 1008 Ile Asn Pro Phe Glu Val Val Tyr Tyr Ala Gly Gly Thr Ser AsnLys 320 325 330 335 tat aga cat ttt gca ggc agt tat gct att caa tgg acaatg att aac 1056 Tyr Arg His Phe Ala Gly Ser Tyr Ala Ile Gln Trp Thr MetIle Asn 340 345 350 tat gca att gat cat ggt att gat aga tac aat ttc tatggt att agc 1104 Tyr Ala Ile Asp His Gly Ile Asp Arg Tyr Asn Phe Tyr GlyIle Ser 355 360 365 ggt aat ttt agt gaa gac gct gaa gat gtt gga gtc attaaa ttt aaa 1152 Gly Asn Phe Ser Glu Asp Ala Glu Asp Val Gly Val Ile LysPhe Lys 370 375 380 aaa ggt ttc aat gca gac gta att gag tat gtt gga gacttt gtg aaa 1200 Lys Gly Phe Asn Ala Asp Val Ile Glu Tyr Val Gly Asp PheVal Lys 385 390 395 cct att aac aaa cct ttg tat tca gtg tat aag aca ctcaaa aag att 1248 Pro Ile Asn Lys Pro Leu Tyr Ser Val Tyr Lys Thr Leu LysLys Ile 400 405 410 415 aaa aaa aga ttt aat taa agaggggaat agacgaatatgaaatttaca 1296 Lys Lys Arg Phe Asn * 420 gagttaaac 1305 41 420 PRTStaphylococcus haemolyticus femA 41 Met Lys Phe Thr Asn Leu Thr Ala ThrGlu Phe Gly Asn Tyr Thr Asp 1 5 10 15 Lys Met Pro Tyr Ser His Phe ThrGln Met Thr Glu Asn Tyr Glu Met 20 25 30 Lys Val Ala Asn Lys Thr Glu ThrHis Leu Val Gly Ile Lys Asn Lys 35 40 45 Asp Asn Glu Val Ile Ala Ala CysMet Leu Thr Ala Val Pro Val Met 50 55 60 Lys Phe Phe Lys Tyr Phe Tyr SerAsn Arg Gly Pro Val Ile Asp Tyr 65 70 75 80 Asp Asn Arg Glu Leu Val HisPhe Phe Phe Asn Glu Leu Thr Lys Tyr 85 90 95 Leu Lys Gln His Asn Cys LeuTyr Val Arg Val Asp Pro Tyr Leu Pro 100 105 110 Tyr Gln Tyr Leu Asn HisAsp Gly Glu Ile Thr Gly Asn Ala Gly Asn 115 120 125 Asp Trp Phe Phe AspLys Met Lys His Leu Gly Phe Glu His Glu Gly 130 135 140 Phe Thr Lys GlyPhe Asp Pro Ile Lys Gln Ile Arg Tyr His Ser Val 145 150 155 160 Leu AspLeu Lys Asn Lys Thr Ser Lys Asp Ile Leu Asn Gly Met Asp 165 170 175 SerLeu Arg Lys Arg Asn Thr Lys Lys Val Gln Lys Asn Gly Val Lys 180 185 190Val Lys Phe Leu Ser Glu Glu Glu Leu Pro Ile Phe Arg Ser Phe Met 195 200205 Glu Asp Thr Thr Glu Thr Lys Glu Phe Gln Asp Arg Asp Asp Ser Phe 210215 220 Tyr Tyr Asn Arg Tyr Arg His Phe Lys Asp His Val Leu Val Pro Leu225 230 235 240 Ala Tyr Ile Lys Phe Asp Glu Tyr Ile Glu Glu Leu Gln AsnGlu Arg 245 250 255 Glu Thr Leu Asn Lys Asp Val Asn Lys Ala Leu Lys AspIle Glu Lys 260 265 270 Arg Pro Asp Asn Lys Lys Ala Phe Asn Lys Lys GluAsn Leu Glu Lys 275 280 285 Gln Leu Asp Ala Asn Gln Gln Lys Leu Asp GluAla Lys Lys Leu Gln 290 295 300 Ala Glu His Gly Asn Glu Leu Pro Ile SerAla Gly Phe Phe Phe Ile 305 310 315 320 Asn Pro Phe Glu Val Val Tyr TyrAla Gly Gly Thr Ser Asn Lys Tyr 325 330 335 Arg His Phe Ala Gly Ser TyrAla Ile Gln Trp Thr Met Ile Asn Tyr 340 345 350 Ala Ile Asp His Gly IleAsp Arg Tyr Asn Phe Tyr Gly Ile Ser Gly 355 360 365 Asn Phe Ser Glu AspAla Glu Asp Val Gly Val Ile Lys Phe Lys Lys 370 375 380 Gly Phe Asn AlaAsp Val Ile Glu Tyr Val Gly Asp Phe Val Lys Pro 385 390 395 400 Ile AsnLys Pro Leu Tyr Ser Val Tyr Lys Thr Leu Lys Lys Ile Lys 405 410 415 LysArg Phe Asn 420 42 1280 DNA Staphylococcus lugdunensis femA CDS(1)...(1242) 42 aca gca aat gaa ttc ggt gat ttc aca gat caa atg cca tatagt cat 48 Thr Ala Asn Glu Phe Gly Asp Phe Thr Asp Gln Met Pro Tyr SerHis 1 5 10 15 ttt act caa atg aca ggt aac tat aat tta aaa gtt gcc gaaaaa aca 96 Phe Thr Gln Met Thr Gly Asn Tyr Asn Leu Lys Val Ala Glu LysThr 20 25 30 gaa aca cat tta gtt ggt gtt aaa aat aat aat aac gaa gta attgca 144 Glu Thr His Leu Val Gly Val Lys Asn Asn Asn Asn Glu Val Ile Ala35 40 45 gca tgt tta ttg aca gct gta cca gtc atg aag ttt ttt aaa tac ttt192 Ala Cys Leu Leu Thr Ala Val Pro Val Met Lys Phe Phe Lys Tyr Phe 5055 60 tac agc aat aga ggc cca gtt ata gat tat gct aac caa gaa ctt gta240 Tyr Ser Asn Arg Gly Pro Val Ile Asp Tyr Ala Asn Gln Glu Leu Val 6570 75 80 cat ttt ttc ttt aat gag cta act aaa tat tta aaa aag tat aac tgt288 His Phe Phe Phe Asn Glu Leu Thr Lys Tyr Leu Lys Lys Tyr Asn Cys 8590 95 ctc tat gtc cgc ata gat cca tac tta cct tat caa tat aga gac cat336 Leu Tyr Val Arg Ile Asp Pro Tyr Leu Pro Tyr Gln Tyr Arg Asp His 100105 110 gac ggt aat ata acg gca aat gct ggc aat gat tgg ttt ttc aat aaa384 Asp Gly Asn Ile Thr Ala Asn Ala Gly Asn Asp Trp Phe Phe Asn Lys 115120 125 atg gaa caa ctc gga tac cat cat gat ggc ttt aca aca gga ttt gat432 Met Glu Gln Leu Gly Tyr His His Asp Gly Phe Thr Thr Gly Phe Asp 130135 140 cca ata tta caa atc aga ttc cat tct att ctt aat tta aag gat aag480 Pro Ile Leu Gln Ile Arg Phe His Ser Ile Leu Asn Leu Lys Asp Lys 145150 155 160 aca gct aaa gat gtt tta aat aat atg gat agt tta cgt aaa agaaat 528 Thr Ala Lys Asp Val Leu Asn Asn Met Asp Ser Leu Arg Lys Arg Asn165 170 175 acc aaa aaa agt tca aaa aat gga gtc aaa gta aag ttc ctt actgaa 576 Thr Lys Lys Ser Ser Lys Asn Gly Val Lys Val Lys Phe Leu Thr Glu180 185 190 gaa gaa cta cct atc ttt cgt tca ttt atg gag cag acg tca gaatct 624 Glu Glu Leu Pro Ile Phe Arg Ser Phe Met Glu Gln Thr Ser Glu Ser195 200 205 aaa gaa ttc tct gat aga gac gac caa ttt tat tac aat cgg tttaag 672 Lys Glu Phe Ser Asp Arg Asp Asp Gln Phe Tyr Tyr Asn Arg Phe Lys210 215 220 tac tat aaa gat agg gtg ctt gtg cct cta gca tat tta aaa tttgat 720 Tyr Tyr Lys Asp Arg Val Leu Val Pro Leu Ala Tyr Leu Lys Phe Asp225 230 235 240 gaa tat ata gaa gaa cta acg aat gaa cga caa act tta gaaaaa gat 768 Glu Tyr Ile Glu Glu Leu Thr Asn Glu Arg Gln Thr Leu Glu LysAsp 245 250 255 tta ggc aaa gca ctt aaa gac att gag aaa cga cca gat aacaaa aaa 816 Leu Gly Lys Ala Leu Lys Asp Ile Glu Lys Arg Pro Asp Asn LysLys 260 265 270 gct tat aat aaa cga gac aac cta caa caa caa ctc gat gccaat caa 864 Ala Tyr Asn Lys Arg Asp Asn Leu Gln Gln Gln Leu Asp Ala AsnGln 275 280 285 caa aag tta aat gag gct aat cag tta caa gcg gaa cac ggtaat gag 912 Gln Lys Leu Asn Glu Ala Asn Gln Leu Gln Ala Glu His Gly AsnGlu 290 295 300 tta cct atc tct gcc ggt ttc ttt att att aat ccg ttt gaagtt gta 960 Leu Pro Ile Ser Ala Gly Phe Phe Ile Ile Asn Pro Phe Glu ValVal 305 310 315 320 tac tac gct gga ggt acc gct aat aaa tat cgt cat tttgca ggt agt 1008 Tyr Tyr Ala Gly Gly Thr Ala Asn Lys Tyr Arg His Phe AlaGly Ser 325 330 335 tac gcg gtt cag tgg act atg att aac tat gct atc gaacac ggc ata 1056 Tyr Ala Val Gln Trp Thr Met Ile Asn Tyr Ala Ile Glu HisGly Ile 340 345 350 gac aga tat aat ttc tac ggc att agt gga aac ttc tcagat gat gct 1104 Asp Arg Tyr Asn Phe Tyr Gly Ile Ser Gly Asn Phe Ser AspAsp Ala 355 360 365 gaa gac gca ggt gtc att cgc ttt aaa aaa ggt tat ggtgca gaa gtg 1152 Glu Asp Ala Gly Val Ile Arg Phe Lys Lys Gly Tyr Gly AlaGlu Val 370 375 380 att gaa tac gtt ggt gat ttt gta aaa cct ata aat aaacct atg tat 1200 Ile Glu Tyr Val Gly Asp Phe Val Lys Pro Ile Asn Lys ProMet Tyr 385 390 395 400 aaa ctt tat tca gtg tta aaa cga att caa aat aagcta tag 1242 Lys Leu Tyr Ser Val Leu Lys Arg Ile Gln Asn Lys Leu * 405410 aggagaatgg attaattatg aaatttacag agtttaac 1280 43 413 PRTStaphylococcus lugdunensis femA 43 Thr Ala Asn Glu Phe Gly Asp Phe ThrAsp Gln Met Pro Tyr Ser His 1 5 10 15 Phe Thr Gln Met Thr Gly Asn TyrAsn Leu Lys Val Ala Glu Lys Thr 20 25 30 Glu Thr His Leu Val Gly Val LysAsn Asn Asn Asn Glu Val Ile Ala 35 40 45 Ala Cys Leu Leu Thr Ala Val ProVal Met Lys Phe Phe Lys Tyr Phe 50 55 60 Tyr Ser Asn Arg Gly Pro Val IleAsp Tyr Ala Asn Gln Glu Leu Val 65 70 75 80 His Phe Phe Phe Asn Glu LeuThr Lys Tyr Leu Lys Lys Tyr Asn Cys 85 90 95 Leu Tyr Val Arg Ile Asp ProTyr Leu Pro Tyr Gln Tyr Arg Asp His 100 105 110 Asp Gly Asn Ile Thr AlaAsn Ala Gly Asn Asp Trp Phe Phe Asn Lys 115 120 125 Met Glu Gln Leu GlyTyr His His Asp Gly Phe Thr Thr Gly Phe Asp 130 135 140 Pro Ile Leu GlnIle Arg Phe His Ser Ile Leu Asn Leu Lys Asp Lys 145 150 155 160 Thr AlaLys Asp Val Leu Asn Asn Met Asp Ser Leu Arg Lys Arg Asn 165 170 175 ThrLys Lys Ser Ser Lys Asn Gly Val Lys Val Lys Phe Leu Thr Glu 180 185 190Glu Glu Leu Pro Ile Phe Arg Ser Phe Met Glu Gln Thr Ser Glu Ser 195 200205 Lys Glu Phe Ser Asp Arg Asp Asp Gln Phe Tyr Tyr Asn Arg Phe Lys 210215 220 Tyr Tyr Lys Asp Arg Val Leu Val Pro Leu Ala Tyr Leu Lys Phe Asp225 230 235 240 Glu Tyr Ile Glu Glu Leu Thr Asn Glu Arg Gln Thr Leu GluLys Asp 245 250 255 Leu Gly Lys Ala Leu Lys Asp Ile Glu Lys Arg Pro AspAsn Lys Lys 260 265 270 Ala Tyr Asn Lys Arg Asp Asn Leu Gln Gln Gln LeuAsp Ala Asn Gln 275 280 285 Gln Lys Leu Asn Glu Ala Asn Gln Leu Gln AlaGlu His Gly Asn Glu 290 295 300 Leu Pro Ile Ser Ala Gly Phe Phe Ile IleAsn Pro Phe Glu Val Val 305 310 315 320 Tyr Tyr Ala Gly Gly Thr Ala AsnLys Tyr Arg His Phe Ala Gly Ser 325 330 335 Tyr Ala Val Gln Trp Thr MetIle Asn Tyr Ala Ile Glu His Gly Ile 340 345 350 Asp Arg Tyr Asn Phe TyrGly Ile Ser Gly Asn Phe Ser Asp Asp Ala 355 360 365 Glu Asp Ala Gly ValIle Arg Phe Lys Lys Gly Tyr Gly Ala Glu Val 370 375 380 Ile Glu Tyr ValGly Asp Phe Val Lys Pro Ile Asn Lys Pro Met Tyr 385 390 395 400 Lys LeuTyr Ser Val Leu Lys Arg Ile Gln Asn Lys Leu 405 410 44 1295 DNAStaphylococcus xylosus femA CDS (1)...(1245) 44 acg caa aag agt ttg ggtgca ttt tca gat aaa atg cca aat agc cat 48 Thr Gln Lys Ser Leu Gly AlaPhe Ser Asp Lys Met Pro Asn Ser His 1 5 10 15 ttc acg caa atg gta gggaat tat gaa ttg aaa att gca gaa agt act 96 Phe Thr Gln Met Val Gly AsnTyr Glu Leu Lys Ile Ala Glu Ser Thr 20 25 30 gaa aca cat tta gta ggt ataaaa aac aat gat aat gaa gtc att gca 144 Glu Thr His Leu Val Gly Ile LysAsn Asn Asp Asn Glu Val Ile Ala 35 40 45 gct tgt tta tta act gca gta ccagta atg aaa ttc ttt aag tat ttt 192 Ala Cys Leu Leu Thr Ala Val Pro ValMet Lys Phe Phe Lys Tyr Phe 50 55 60 tat act aat aga ggt ccg gtt ata gatttt gaa aat aaa gaa tta gtg 240 Tyr Thr Asn Arg Gly Pro Val Ile Asp PheGlu Asn Lys Glu Leu Val 65 70 75 80 cat tac ttt ttc aat gaa cta tct aaatat gtg aaa aaa cat aat gcg 288 His Tyr Phe Phe Asn Glu Leu Ser Lys TyrVal Lys Lys His Asn Ala 85 90 95 ctt tat tta aga gtt gat cct tat tta gcatat caa tac cgt aat cat 336 Leu Tyr Leu Arg Val Asp Pro Tyr Leu Ala TyrGln Tyr Arg Asn His 100 105 110 gat ggt gag gta ttg gaa aat gca gga catgat tgg att ttc gat aaa 384 Asp Gly Glu Val Leu Glu Asn Ala Gly His AspTrp Ile Phe Asp Lys 115 120 125 atg aag cag ctt gga tat aaa cac caa ggattt tta act ggt ttc gat 432 Met Lys Gln Leu Gly Tyr Lys His Gln Gly PheLeu Thr Gly Phe Asp 130 135 140 tca att att caa att agg ttc cac tct gtactg gat tta gta ggt aaa 480 Ser Ile Ile Gln Ile Arg Phe His Ser Val LeuAsp Leu Val Gly Lys 145 150 155 160 act gct aaa gat gta cta aat ggt atggat agt tta cgt aaa cgt aat 528 Thr Ala Lys Asp Val Leu Asn Gly Met AspSer Leu Arg Lys Arg Asn 165 170 175 act aaa aaa gta caa aaa aat ggc gtgaaa gta agg ttc tta agg gaa 576 Thr Lys Lys Val Gln Lys Asn Gly Val LysVal Arg Phe Leu Arg Glu 180 185 190 gat gag ttg cca att ttc cgt tca ttcatg gaa gat aca tct gaa act 624 Asp Glu Leu Pro Ile Phe Arg Ser Phe MetGlu Asp Thr Ser Glu Thr 195 200 205 aaa gac ttt gac gat aga gac gat ggcttt tac tac aat aga tta agg 672 Lys Asp Phe Asp Asp Arg Asp Asp Gly PheTyr Tyr Asn Arg Leu Arg 210 215 220 tat tat aaa gat cgc gta tta gta cctcta gct tat atg gat ttc aat 720 Tyr Tyr Lys Asp Arg Val Leu Val Pro LeuAla Tyr Met Asp Phe Asn 225 230 235 240 gaa tat att gaa gaa ttg caa gctgaa cgt gag gtg tta agc aaa gat 768 Glu Tyr Ile Glu Glu Leu Gln Ala GluArg Glu Val Leu Ser Lys Asp 245 250 255 atc aat aaa gca gta aaa gat atcgag aaa aga cct gaa aat aaa aaa 816 Ile Asn Lys Ala Val Lys Asp Ile GluLys Arg Pro Glu Asn Lys Lys 260 265 270 gca tat aat aaa aaa gat aat ctagag aaa caa ctt ata gcg aat caa 864 Ala Tyr Asn Lys Lys Asp Asn Leu GluLys Gln Leu Ile Ala Asn Gln 275 280 285 caa aaa att gat gaa gct aaa actcta caa gag aag cat ggt aac gaa 912 Gln Lys Ile Asp Glu Ala Lys Thr LeuGln Glu Lys His Gly Asn Glu 290 295 300 cta cca atc tca gca gca tat ttcatc att aac cct tat gaa gta gtg 960 Leu Pro Ile Ser Ala Ala Tyr Phe IleIle Asn Pro Tyr Glu Val Val 305 310 315 320 tat tat gcg ggt gga acg tcaaat gag ttt aga cat ttt gct ggt agt 1008 Tyr Tyr Ala Gly Gly Thr Ser AsnGlu Phe Arg His Phe Ala Gly Ser 325 330 335 tat gcc att caa tgg aag atgatt aac tat gct att gac cat aat att 1056 Tyr Ala Ile Gln Trp Lys Met IleAsn Tyr Ala Ile Asp His Asn Ile 340 345 350 gat aga tat aat ttt tat ggaatt agt ggt cat ttt aca gaa gat gca 1104 Asp Arg Tyr Asn Phe Tyr Gly IleSer Gly His Phe Thr Glu Asp Ala 355 360 365 gaa gat gcc ggt gta gtt aaattt aaa aaa gga ttt aat gcg gat gta 1152 Glu Asp Ala Gly Val Val Lys PheLys Lys Gly Phe Asn Ala Asp Val 370 375 380 gtg gaa tat gtt ggt gat tttatt aaa cca atc aat aaa cca atg tac 1200 Val Glu Tyr Val Gly Asp Phe IleLys Pro Ile Asn Lys Pro Met Tyr 385 390 395 400 aaa att tat acg aca ttaaag aaa att aaa gat aaa aag aaa taa 1245 Lys Ile Tyr Thr Thr Leu Lys LysIle Lys Asp Lys Lys Lys * 405 410 acatttaata gaagggaact aagctagaatgaaatttaca gagttaaacc 1295 45 414 PRT Staphylococcus xylosus femA 45 ThrGln Lys Ser Leu Gly Ala Phe Ser Asp Lys Met Pro Asn Ser His 1 5 10 15Phe Thr Gln Met Val Gly Asn Tyr Glu Leu Lys Ile Ala Glu Ser Thr 20 25 30Glu Thr His Leu Val Gly Ile Lys Asn Asn Asp Asn Glu Val Ile Ala 35 40 45Ala Cys Leu Leu Thr Ala Val Pro Val Met Lys Phe Phe Lys Tyr Phe 50 55 60Tyr Thr Asn Arg Gly Pro Val Ile Asp Phe Glu Asn Lys Glu Leu Val 65 70 7580 His Tyr Phe Phe Asn Glu Leu Ser Lys Tyr Val Lys Lys His Asn Ala 85 9095 Leu Tyr Leu Arg Val Asp Pro Tyr Leu Ala Tyr Gln Tyr Arg Asn His 100105 110 Asp Gly Glu Val Leu Glu Asn Ala Gly His Asp Trp Ile Phe Asp Lys115 120 125 Met Lys Gln Leu Gly Tyr Lys His Gln Gly Phe Leu Thr Gly PheAsp 130 135 140 Ser Ile Ile Gln Ile Arg Phe His Ser Val Leu Asp Leu ValGly Lys 145 150 155 160 Thr Ala Lys Asp Val Leu Asn Gly Met Asp Ser LeuArg Lys Arg Asn 165 170 175 Thr Lys Lys Val Gln Lys Asn Gly Val Lys ValArg Phe Leu Arg Glu 180 185 190 Asp Glu Leu Pro Ile Phe Arg Ser Phe MetGlu Asp Thr Ser Glu Thr 195 200 205 Lys Asp Phe Asp Asp Arg Asp Asp GlyPhe Tyr Tyr Asn Arg Leu Arg 210 215 220 Tyr Tyr Lys Asp Arg Val Leu ValPro Leu Ala Tyr Met Asp Phe Asn 225 230 235 240 Glu Tyr Ile Glu Glu LeuGln Ala Glu Arg Glu Val Leu Ser Lys Asp 245 250 255 Ile Asn Lys Ala ValLys Asp Ile Glu Lys Arg Pro Glu Asn Lys Lys 260 265 270 Ala Tyr Asn LysLys Asp Asn Leu Glu Lys Gln Leu Ile Ala Asn Gln 275 280 285 Gln Lys IleAsp Glu Ala Lys Thr Leu Gln Glu Lys His Gly Asn Glu 290 295 300 Leu ProIle Ser Ala Ala Tyr Phe Ile Ile Asn Pro Tyr Glu Val Val 305 310 315 320Tyr Tyr Ala Gly Gly Thr Ser Asn Glu Phe Arg His Phe Ala Gly Ser 325 330335 Tyr Ala Ile Gln Trp Lys Met Ile Asn Tyr Ala Ile Asp His Asn Ile 340345 350 Asp Arg Tyr Asn Phe Tyr Gly Ile Ser Gly His Phe Thr Glu Asp Ala355 360 365 Glu Asp Ala Gly Val Val Lys Phe Lys Lys Gly Phe Asn Ala AspVal 370 375 380 Val Glu Tyr Val Gly Asp Phe Ile Lys Pro Ile Asn Lys ProMet Tyr 385 390 395 400 Lys Ile Tyr Thr Thr Leu Lys Lys Ile Lys Asp LysLys Lys 405 410 46 1283 DNA Staphylococcus capitis femA CDS (1)...(1236)46 aca gct aaa gaa ttt agt gac ttt act gat caa atg cct tat agc cat 48Thr Ala Lys Glu Phe Ser Asp Phe Thr Asp Gln Met Pro Tyr Ser His 1 5 1015 ttt act cag atg gaa ggt aat tat gaa ctt aaa gtt gct gaa ggt acg 96Phe Thr Gln Met Glu Gly Asn Tyr Glu Leu Lys Val Ala Glu Gly Thr 20 25 30gat tca cat ctc gta gga att aaa aat aat gac aac caa gtg att gca 144 AspSer His Leu Val Gly Ile Lys Asn Asn Asp Asn Gln Val Ile Ala 35 40 45 gcatgt tta tta act gct gta cct gta atg aaa att ttt aaa tat ttt 192 Ala CysLeu Leu Thr Ala Val Pro Val Met Lys Ile Phe Lys Tyr Phe 50 55 60 tac tcaaat cgc ggg cca gtg att gat tat gat aat aaa gag ctt gtt 240 Tyr Ser AsnArg Gly Pro Val Ile Asp Tyr Asp Asn Lys Glu Leu Val 65 70 75 80 cac tttttc ttt aat gaa tta agt aaa tat gta aaa aag cat aat tgt 288 His Phe PhePhe Asn Glu Leu Ser Lys Tyr Val Lys Lys His Asn Cys 85 90 95 ctt tat ctaaga gtt gac cct tat ctt cct tat caa tac tta aat cat 336 Leu Tyr Leu ArgVal Asp Pro Tyr Leu Pro Tyr Gln Tyr Leu Asn His 100 105 110 gac ggt gaaatt att gga aat gct ggc cat gat tgg ttt ttc aat aag 384 Asp Gly Glu IleIle Gly Asn Ala Gly His Asp Trp Phe Phe Asn Lys 115 120 125 atg gaa gaatta gga ttt gaa cat gaa ggc ttt cat aaa ggc ttc cat 432 Met Glu Glu LeuGly Phe Glu His Glu Gly Phe His Lys Gly Phe His 130 135 140 cct atc ttacaa gta aga tat cat tca gtt tta gat tta aaa gat aaa 480 Pro Ile Leu GlnVal Arg Tyr His Ser Val Leu Asp Leu Lys Asp Lys 145 150 155 160 acg gctaaa gat gta ctc aaa gga atg gat agt tta aga aag cgt aat 528 Thr Ala LysAsp Val Leu Lys Gly Met Asp Ser Leu Arg Lys Arg Asn 165 170 175 act aagaaa gta caa aaa aat ggt gtc aaa gtc cgt ttc cta tcc gaa 576 Thr Lys LysVal Gln Lys Asn Gly Val Lys Val Arg Phe Leu Ser Glu 180 185 190 gat gaatta cct atc ttt aga tca ttt atg gaa gat act aca gaa acg 624 Asp Glu LeuPro Ile Phe Arg Ser Phe Met Glu Asp Thr Thr Glu Thr 195 200 205 aaa gagttc gcc gat aga gat gat agt ttc tat tat aat cga tta aaa 672 Lys Glu PheAla Asp Arg Asp Asp Ser Phe Tyr Tyr Asn Arg Leu Lys 210 215 220 tac tttaaa gat aga gta tta gta cca tta gca tat gtt gac ttc gat 720 Tyr Phe LysAsp Arg Val Leu Val Pro Leu Ala Tyr Val Asp Phe Asp 225 230 235 240 gagtat att gaa gaa ctt aat aat gaa aga gat gtt ctt aat aaa gat 768 Glu TyrIle Glu Glu Leu Asn Asn Glu Arg Asp Val Leu Asn Lys Asp 245 250 255 ttaaat aag gcg ctc aaa gat att gag aag aga cct gat aat aag aaa 816 Leu AsnLys Ala Leu Lys Asp Ile Glu Lys Arg Pro Asp Asn Lys Lys 260 265 270 gcttat aac aaa aga gat aat ctt caa caa caa tta gat gca aat caa 864 Ala TyrAsn Lys Arg Asp Asn Leu Gln Gln Gln Leu Asp Ala Asn Gln 275 280 285 caaaaa att gat gaa gct aaa aac tta caa caa gaa cat ggt aat gaa 912 Gln LysIle Asp Glu Ala Lys Asn Leu Gln Gln Glu His Gly Asn Glu 290 295 300 ttacct att tca gct gga tat ttc ttc att aat ccg ttt gaa gtt gtt 960 Leu ProIle Ser Ala Gly Tyr Phe Phe Ile Asn Pro Phe Glu Val Val 305 310 315 320tat tac gca ggt ggc aca tcg aat cgt tat cgt cac tat gcc gga agt 1008 TyrTyr Ala Gly Gly Thr Ser Asn Arg Tyr Arg His Tyr Ala Gly Ser 325 330 335tat gca att caa tgg aaa atg ata aac tat gct tta gaa cat gga att 1056 TyrAla Ile Gln Trp Lys Met Ile Asn Tyr Ala Leu Glu His Gly Ile 340 345 350aac cgt tat aat ttt tat gga gtt agt ggg gac ttc agt gaa gac gct 1104 AsnArg Tyr Asn Phe Tyr Gly Val Ser Gly Asp Phe Ser Glu Asp Ala 355 360 365gaa gat gta gga gta att aag ttc aaa aaa ggc tat aat gct gat gtt 1152 GluAsp Val Gly Val Ile Lys Phe Lys Lys Gly Tyr Asn Ala Asp Val 370 375 380att gaa tat gta ggt gat ttt atc aag cca atc aat aaa cct atg tat 1200 IleGlu Tyr Val Gly Asp Phe Ile Lys Pro Ile Asn Lys Pro Met Tyr 385 390 395400 gca atc tat aac gca ctt aaa aag tta aag aaa tag atttttttac 1246 AlaIle Tyr Asn Ala Leu Lys Lys Leu Lys Lys * 405 410 caacccaatt atctaattatgaaatttaca gagttaa 1283 47 411 PRT Staphylococcus capitis femA 47 ThrAla Lys Glu Phe Ser Asp Phe Thr Asp Gln Met Pro Tyr Ser His 1 5 10 15Phe Thr Gln Met Glu Gly Asn Tyr Glu Leu Lys Val Ala Glu Gly Thr 20 25 30Asp Ser His Leu Val Gly Ile Lys Asn Asn Asp Asn Gln Val Ile Ala 35 40 45Ala Cys Leu Leu Thr Ala Val Pro Val Met Lys Ile Phe Lys Tyr Phe 50 55 60Tyr Ser Asn Arg Gly Pro Val Ile Asp Tyr Asp Asn Lys Glu Leu Val 65 70 7580 His Phe Phe Phe Asn Glu Leu Ser Lys Tyr Val Lys Lys His Asn Cys 85 9095 Leu Tyr Leu Arg Val Asp Pro Tyr Leu Pro Tyr Gln Tyr Leu Asn His 100105 110 Asp Gly Glu Ile Ile Gly Asn Ala Gly His Asp Trp Phe Phe Asn Lys115 120 125 Met Glu Glu Leu Gly Phe Glu His Glu Gly Phe His Lys Gly PheHis 130 135 140 Pro Ile Leu Gln Val Arg Tyr His Ser Val Leu Asp Leu LysAsp Lys 145 150 155 160 Thr Ala Lys Asp Val Leu Lys Gly Met Asp Ser LeuArg Lys Arg Asn 165 170 175 Thr Lys Lys Val Gln Lys Asn Gly Val Lys ValArg Phe Leu Ser Glu 180 185 190 Asp Glu Leu Pro Ile Phe Arg Ser Phe MetGlu Asp Thr Thr Glu Thr 195 200 205 Lys Glu Phe Ala Asp Arg Asp Asp SerPhe Tyr Tyr Asn Arg Leu Lys 210 215 220 Tyr Phe Lys Asp Arg Val Leu ValPro Leu Ala Tyr Val Asp Phe Asp 225 230 235 240 Glu Tyr Ile Glu Glu LeuAsn Asn Glu Arg Asp Val Leu Asn Lys Asp 245 250 255 Leu Asn Lys Ala LeuLys Asp Ile Glu Lys Arg Pro Asp Asn Lys Lys 260 265 270 Ala Tyr Asn LysArg Asp Asn Leu Gln Gln Gln Leu Asp Ala Asn Gln 275 280 285 Gln Lys IleAsp Glu Ala Lys Asn Leu Gln Gln Glu His Gly Asn Glu 290 295 300 Leu ProIle Ser Ala Gly Tyr Phe Phe Ile Asn Pro Phe Glu Val Val 305 310 315 320Tyr Tyr Ala Gly Gly Thr Ser Asn Arg Tyr Arg His Tyr Ala Gly Ser 325 330335 Tyr Ala Ile Gln Trp Lys Met Ile Asn Tyr Ala Leu Glu His Gly Ile 340345 350 Asn Arg Tyr Asn Phe Tyr Gly Val Ser Gly Asp Phe Ser Glu Asp Ala355 360 365 Glu Asp Val Gly Val Ile Lys Phe Lys Lys Gly Tyr Asn Ala AspVal 370 375 380 Ile Glu Tyr Val Gly Asp Phe Ile Lys Pro Ile Asn Lys ProMet Tyr 385 390 395 400 Ala Ile Tyr Asn Ala Leu Lys Lys Leu Lys Lys 405410 48 1297 DNA Staphylococcus schleiferi femA CDS (1)...(1248) 48 acgacg gct gaa ttt ggt gcg ttt aca gat caa atg cca tat agc cat 48 Thr ThrAla Glu Phe Gly Ala Phe Thr Asp Gln Met Pro Tyr Ser His 1 5 10 15 ttcacg caa atg gta ggg aac tat gaa tta aag gtt gct gaa ggt gtt 96 Phe ThrGln Met Val Gly Asn Tyr Glu Leu Lys Val Ala Glu Gly Val 20 25 30 gaa acacat ctt gtc ggc att aaa gat aac aac aat aac gta cta gca 144 Glu Thr HisLeu Val Gly Ile Lys Asp Asn Asn Asn Asn Val Leu Ala 35 40 45 gca tgt ttactg aca gca gtg cca gta atg aag ttt ttt aaa tat ttt 192 Ala Cys Leu LeuThr Ala Val Pro Val Met Lys Phe Phe Lys Tyr Phe 50 55 60 tat tca aac cgcgga cca gtc atg gac tac gaa aat aaa gag ctc gtt 240 Tyr Ser Asn Arg GlyPro Val Met Asp Tyr Glu Asn Lys Glu Leu Val 65 70 75 80 cat ttc ttt tttaat gaa ctt tca aaa tat gtt aag aaa tat cac gca 288 His Phe Phe Phe AsnGlu Leu Ser Lys Tyr Val Lys Lys Tyr His Ala 85 90 95 ttg tat ttg aga gtagac cct tat tta cca atg tta aag cga aac cat 336 Leu Tyr Leu Arg Val AspPro Tyr Leu Pro Met Leu Lys Arg Asn His 100 105 110 gat ggt gaa gtg attgaa aga tac ggc agt gac tgg ttt ttt gat aaa 384 Asp Gly Glu Val Ile GluArg Tyr Gly Ser Asp Trp Phe Phe Asp Lys 115 120 125 atg gct gaa tta aacttt gaa cat gaa ggt ttc aca act ggg ttt gat 432 Met Ala Glu Leu Asn PheGlu His Glu Gly Phe Thr Thr Gly Phe Asp 130 135 140 aca ata agg caa attcgt ttt cat tct gtg ctc gat gtt gaa aat aaa 480 Thr Ile Arg Gln Ile ArgPhe His Ser Val Leu Asp Val Glu Asn Lys 145 150 155 160 aca tca aaa gacatc tta aat caa atg gat aat tta agg aaa aga aat 528 Thr Ser Lys Asp IleLeu Asn Gln Met Asp Asn Leu Arg Lys Arg Asn 165 170 175 acg aaa aaa gtacag aaa aat ggt gtg aaa gtc cgc tat cta aac gaa 576 Thr Lys Lys Val GlnLys Asn Gly Val Lys Val Arg Tyr Leu Asn Glu 180 185 190 gat gaa tta catatt ttc cgt tcg ttt atg gaa gat aca tct gaa aca 624 Asp Glu Leu His IlePhe Arg Ser Phe Met Glu Asp Thr Ser Glu Thr 195 200 205 aaa gat ttt gtagat aga gat gac gat ttt tat tat cat cgt atg aaa 672 Lys Asp Phe Val AspArg Asp Asp Asp Phe Tyr Tyr His Arg Met Lys 210 215 220 tac tat aaa gatcgt gtc cgc gta cca cta gcg tat att gat ttt aat 720 Tyr Tyr Lys Asp ArgVal Arg Val Pro Leu Ala Tyr Ile Asp Phe Asn 225 230 235 240 gca tat ttagca gag ctc aac act gaa gcg caa gac ttt aaa aaa gaa 768 Ala Tyr Leu AlaGlu Leu Asn Thr Glu Ala Gln Asp Phe Lys Lys Glu 245 250 255 att gca aaagca gat aaa gac atc gac aag cgt cct gaa aat cag aaa 816 Ile Ala Lys AlaAsp Lys Asp Ile Asp Lys Arg Pro Glu Asn Gln Lys 260 265 270 gcc ata aataaa aag aaa aat tta gag caa caa cta gaa gcg aat caa 864 Ala Ile Asn LysLys Lys Asn Leu Glu Gln Gln Leu Glu Ala Asn Gln 275 280 285 gct aaa ataaaa gaa gca gaa aca ttg caa ctt aaa cac ggt gac aca 912 Ala Lys Ile LysGlu Ala Glu Thr Leu Gln Leu Lys His Gly Asp Thr 290 295 300 tta ccg atttcg gct gga ttc ttt att att aat cca ttt gag gtt gtt 960 Leu Pro Ile SerAla Gly Phe Phe Ile Ile Asn Pro Phe Glu Val Val 305 310 315 320 tat tatgca ggc ggc aca gca aac gaa ttt cgt cat ttt gct gga agc 1008 Tyr Tyr AlaGly Gly Thr Ala Asn Glu Phe Arg His Phe Ala Gly Ser 325 330 335 tac gcagtg caa tgg gaa atg att aat tat gcg att gat tat caa att 1056 Tyr Ala ValGln Trp Glu Met Ile Asn Tyr Ala Ile Asp Tyr Gln Ile 340 345 350 cca agatat aac ttt tat ggc att agt ggt gat ttt tca gaa gat gca 1104 Pro Arg TyrAsn Phe Tyr Gly Ile Ser Gly Asp Phe Ser Glu Asp Ala 355 360 365 gaa gatgca ggt gtg ata aaa ttt aaa aaa ggc tat aat gca gaa gta 1152 Glu Asp AlaGly Val Ile Lys Phe Lys Lys Gly Tyr Asn Ala Glu Val 370 375 380 ata gaatat gtc ggt gat ttt att aag cct ata aac aaa cct gcc tat 1200 Ile Glu TyrVal Gly Asp Phe Ile Lys Pro Ile Asn Lys Pro Ala Tyr 385 390 395 400 acagtc tac tta aaa tta aag caa tta aaa gac aag ata aaa aga taa 1248 Thr ValTyr Leu Lys Leu Lys Gln Leu Lys Asp Lys Ile Lys Arg * 405 410 415gatatagcaa agagaagggg atttattggt atgaaattta cagagttaa 1297 49 415 PRTStaphylococcus schleiferi femA 49 Thr Thr Ala Glu Phe Gly Ala Phe ThrAsp Gln Met Pro Tyr Ser His 1 5 10 15 Phe Thr Gln Met Val Gly Asn TyrGlu Leu Lys Val Ala Glu Gly Val 20 25 30 Glu Thr His Leu Val Gly Ile LysAsp Asn Asn Asn Asn Val Leu Ala 35 40 45 Ala Cys Leu Leu Thr Ala Val ProVal Met Lys Phe Phe Lys Tyr Phe 50 55 60 Tyr Ser Asn Arg Gly Pro Val MetAsp Tyr Glu Asn Lys Glu Leu Val 65 70 75 80 His Phe Phe Phe Asn Glu LeuSer Lys Tyr Val Lys Lys Tyr His Ala 85 90 95 Leu Tyr Leu Arg Val Asp ProTyr Leu Pro Met Leu Lys Arg Asn His 100 105 110 Asp Gly Glu Val Ile GluArg Tyr Gly Ser Asp Trp Phe Phe Asp Lys 115 120 125 Met Ala Glu Leu AsnPhe Glu His Glu Gly Phe Thr Thr Gly Phe Asp 130 135 140 Thr Ile Arg GlnIle Arg Phe His Ser Val Leu Asp Val Glu Asn Lys 145 150 155 160 Thr SerLys Asp Ile Leu Asn Gln Met Asp Asn Leu Arg Lys Arg Asn 165 170 175 ThrLys Lys Val Gln Lys Asn Gly Val Lys Val Arg Tyr Leu Asn Glu 180 185 190Asp Glu Leu His Ile Phe Arg Ser Phe Met Glu Asp Thr Ser Glu Thr 195 200205 Lys Asp Phe Val Asp Arg Asp Asp Asp Phe Tyr Tyr His Arg Met Lys 210215 220 Tyr Tyr Lys Asp Arg Val Arg Val Pro Leu Ala Tyr Ile Asp Phe Asn225 230 235 240 Ala Tyr Leu Ala Glu Leu Asn Thr Glu Ala Gln Asp Phe LysLys Glu 245 250 255 Ile Ala Lys Ala Asp Lys Asp Ile Asp Lys Arg Pro GluAsn Gln Lys 260 265 270 Ala Ile Asn Lys Lys Lys Asn Leu Glu Gln Gln LeuGlu Ala Asn Gln 275 280 285 Ala Lys Ile Lys Glu Ala Glu Thr Leu Gln LeuLys His Gly Asp Thr 290 295 300 Leu Pro Ile Ser Ala Gly Phe Phe Ile IleAsn Pro Phe Glu Val Val 305 310 315 320 Tyr Tyr Ala Gly Gly Thr Ala AsnGlu Phe Arg His Phe Ala Gly Ser 325 330 335 Tyr Ala Val Gln Trp Glu MetIle Asn Tyr Ala Ile Asp Tyr Gln Ile 340 345 350 Pro Arg Tyr Asn Phe TyrGly Ile Ser Gly Asp Phe Ser Glu Asp Ala 355 360 365 Glu Asp Ala Gly ValIle Lys Phe Lys Lys Gly Tyr Asn Ala Glu Val 370 375 380 Ile Glu Tyr ValGly Asp Phe Ile Lys Pro Ile Asn Lys Pro Ala Tyr 385 390 395 400 Thr ValTyr Leu Lys Leu Lys Gln Leu Lys Asp Lys Ile Lys Arg 405 410 415 50 1284DNA Staphylococcus sciuri femA CDS (1)...(1233) 50 aca ctg gaa ttt gaagct ttt aca aat aaa atg ccg tac gcg cat ttt 48 Thr Leu Glu Phe Glu AlaPhe Thr Asn Lys Met Pro Tyr Ala His Phe 1 5 10 15 aca caa gca gta ggtaat tat gaa tta aaa aca tct gaa ggt act tca 96 Thr Gln Ala Val Gly AsnTyr Glu Leu Lys Thr Ser Glu Gly Thr Ser 20 25 30 aca cat tta gta ggg gtcaaa gat aat caa ggt gaa gta tta gct gcg 144 Thr His Leu Val Gly Val LysAsp Asn Gln Gly Glu Val Leu Ala Ala 35 40 45 tgt ctg tta aca agt gta ccagtt atg aag aaa ttt aat tac ttt tac 192 Cys Leu Leu Thr Ser Val Pro ValMet Lys Lys Phe Asn Tyr Phe Tyr 50 55 60 tca aat aga gga cca gta atg gattat gac aac aaa gaa ctt gtt gac 240 Ser Asn Arg Gly Pro Val Met Asp TyrAsp Asn Lys Glu Leu Val Asp 65 70 75 80 ttt ttc ttt aaa gaa atc gtg agctat tta aaa agt tat aaa gga tta 288 Phe Phe Phe Lys Glu Ile Val Ser TyrLeu Lys Ser Tyr Lys Gly Leu 85 90 95 ttc ttt aga atc gat cct tac ttg ccatat caa cta aga gat cat gat 336 Phe Phe Arg Ile Asp Pro Tyr Leu Pro TyrGln Leu Arg Asp His Asp 100 105 110 ggc aat att aaa aaa tca ttc aac cgtgat ggt tta att aaa caa ttt 384 Gly Asn Ile Lys Lys Ser Phe Asn Arg AspGly Leu Ile Lys Gln Phe 115 120 125 gaa tca tta ggt tat gaa cac caa ggcttc aca act ggt ttc cac cca 432 Glu Ser Leu Gly Tyr Glu His Gln Gly PheThr Thr Gly Phe His Pro 130 135 140 ata cat caa att aga tgg cat tct gtactt gat tta gaa agt atg gac 480 Ile His Gln Ile Arg Trp His Ser Val LeuAsp Leu Glu Ser Met Asp 145 150 155 160 gaa aag acg ctc atc aag aac atggac agt tta aga aaa aga aat act 528 Glu Lys Thr Leu Ile Lys Asn Met AspSer Leu Arg Lys Arg Asn Thr 165 170 175 aaa aaa gtt caa aaa aat ggt gttaaa gtt cgt ttt cta tct aaa gat 576 Lys Lys Val Gln Lys Asn Gly Val LysVal Arg Phe Leu Ser Lys Asp 180 185 190 gaa atg ccg ata ttc cgt caa tttatg gaa gat act aca gag aag aaa 624 Glu Met Pro Ile Phe Arg Gln Phe MetGlu Asp Thr Thr Glu Lys Lys 195 200 205 gat ttc aac gat cgt ggc gat gacttc tat tac aat aga tta aaa tac 672 Asp Phe Asn Asp Arg Gly Asp Asp PheTyr Tyr Asn Arg Leu Lys Tyr 210 215 220 ttt gaa aat gta aag att cct ttagca tat ata gac ttt gaa act tac 720 Phe Glu Asn Val Lys Ile Pro Leu AlaTyr Ile Asp Phe Glu Thr Tyr 225 230 235 240 att cca caa tta gaa aaa gaacat gaa caa tac aac aaa gat att gca 768 Ile Pro Gln Leu Glu Lys Glu HisGlu Gln Tyr Asn Lys Asp Ile Ala 245 250 255 aaa gct gaa aaa gat tta gaaaag aaa cca gat aat caa aaa acg att 816 Lys Ala Glu Lys Asp Leu Glu LysLys Pro Asp Asn Gln Lys Thr Ile 260 265 270 aat aaa ata gac aac tta aaacaa caa aga gaa gca aat gaa gct aaa 864 Asn Lys Ile Asp Asn Leu Lys GlnGln Arg Glu Ala Asn Glu Ala Lys 275 280 285 tta gaa gaa gca ctt caa ctacaa caa gaa cat ggt gat aca tta cca 912 Leu Glu Glu Ala Leu Gln Leu GlnGln Glu His Gly Asp Thr Leu Pro 290 295 300 ata gca gct ggt ttc ttt attatt aat cca ttt gaa gtt gta tat tat 960 Ile Ala Ala Gly Phe Phe Ile IleAsn Pro Phe Glu Val Val Tyr Tyr 305 310 315 320 gca ggt ggt tca tcg aatgaa tat cgt cac ttt gca ggt agt tat gca 1008 Ala Gly Gly Ser Ser Asn GluTyr Arg His Phe Ala Gly Ser Tyr Ala 325 330 335 att cag tgg gaa atg attaaa tac gcg tta gat cac aac att gac cgt 1056 Ile Gln Trp Glu Met Ile LysTyr Ala Leu Asp His Asn Ile Asp Arg 340 345 350 tat aac ttc tat ggt atcagc gga gac ttc tca gaa gat gca cct gat 1104 Tyr Asn Phe Tyr Gly Ile SerGly Asp Phe Ser Glu Asp Ala Pro Asp 355 360 365 gtt ggc gtt att aaa tttaaa aaa ggt tac aat gca gat gtt tat gaa 1152 Val Gly Val Ile Lys Phe LysLys Gly Tyr Asn Ala Asp Val Tyr Glu 370 375 380 tat att ggt gat ttc gttaaa cca att aat aaa cca gcg tac aaa gca 1200 Tyr Ile Gly Asp Phe Val LysPro Ile Asn Lys Pro Ala Tyr Lys Ala 385 390 395 400 tat aca aca cta aaaaaa gta tta aaa aaa taa atgattttca gtaagagagg 1253 Tyr Thr Thr Leu LysLys Val Leu Lys Lys * 405 410 aatttagata atatgaaatt tacagagtta a 1284 51410 PRT Staphylococcus sciuri femA 51 Thr Leu Glu Phe Glu Ala Phe ThrAsn Lys Met Pro Tyr Ala His Phe 1 5 10 15 Thr Gln Ala Val Gly Asn TyrGlu Leu Lys Thr Ser Glu Gly Thr Ser 20 25 30 Thr His Leu Val Gly Val LysAsp Asn Gln Gly Glu Val Leu Ala Ala 35 40 45 Cys Leu Leu Thr Ser Val ProVal Met Lys Lys Phe Asn Tyr Phe Tyr 50 55 60 Ser Asn Arg Gly Pro Val MetAsp Tyr Asp Asn Lys Glu Leu Val Asp 65 70 75 80 Phe Phe Phe Lys Glu IleVal Ser Tyr Leu Lys Ser Tyr Lys Gly Leu 85 90 95 Phe Phe Arg Ile Asp ProTyr Leu Pro Tyr Gln Leu Arg Asp His Asp 100 105 110 Gly Asn Ile Lys LysSer Phe Asn Arg Asp Gly Leu Ile Lys Gln Phe 115 120 125 Glu Ser Leu GlyTyr Glu His Gln Gly Phe Thr Thr Gly Phe His Pro 130 135 140 Ile His GlnIle Arg Trp His Ser Val Leu Asp Leu Glu Ser Met Asp 145 150 155 160 GluLys Thr Leu Ile Lys Asn Met Asp Ser Leu Arg Lys Arg Asn Thr 165 170 175Lys Lys Val Gln Lys Asn Gly Val Lys Val Arg Phe Leu Ser Lys Asp 180 185190 Glu Met Pro Ile Phe Arg Gln Phe Met Glu Asp Thr Thr Glu Lys Lys 195200 205 Asp Phe Asn Asp Arg Gly Asp Asp Phe Tyr Tyr Asn Arg Leu Lys Tyr210 215 220 Phe Glu Asn Val Lys Ile Pro Leu Ala Tyr Ile Asp Phe Glu ThrTyr 225 230 235 240 Ile Pro Gln Leu Glu Lys Glu His Glu Gln Tyr Asn LysAsp Ile Ala 245 250 255 Lys Ala Glu Lys Asp Leu Glu Lys Lys Pro Asp AsnGln Lys Thr Ile 260 265 270 Asn Lys Ile Asp Asn Leu Lys Gln Gln Arg GluAla Asn Glu Ala Lys 275 280 285 Leu Glu Glu Ala Leu Gln Leu Gln Gln GluHis Gly Asp Thr Leu Pro 290 295 300 Ile Ala Ala Gly Phe Phe Ile Ile AsnPro Phe Glu Val Val Tyr Tyr 305 310 315 320 Ala Gly Gly Ser Ser Asn GluTyr Arg His Phe Ala Gly Ser Tyr Ala 325 330 335 Ile Gln Trp Glu Met IleLys Tyr Ala Leu Asp His Asn Ile Asp Arg 340 345 350 Tyr Asn Phe Tyr GlyIle Ser Gly Asp Phe Ser Glu Asp Ala Pro Asp 355 360 365 Val Gly Val IleLys Phe Lys Lys Gly Tyr Asn Ala Asp Val Tyr Glu 370 375 380 Tyr Ile GlyAsp Phe Val Lys Pro Ile Asn Lys Pro Ala Tyr Lys Ala 385 390 395 400 TyrThr Thr Leu Lys Lys Val Leu Lys Lys 405 410 52 1343 DNA Staphylococcushominis femA CDS (64)...(1317) 52 taaaatttta aaattagtca actcaaattaaataaagatt ctaaattagg agttatagag 60 ata atg aag ttt aca aat tta aca gctaca gaa ttt ggc gat ttt act 108 Met Lys Phe Thr Asn Leu Thr Ala Thr GluPhe Gly Asp Phe Thr 1 5 10 15 gaa aaa atg cca tat agc cat ttt aca cagatg act gaa aat tat gag 156 Glu Lys Met Pro Tyr Ser His Phe Thr Gln MetThr Glu Asn Tyr Glu 20 25 30 tta aaa gtt gct gag aaa act gaa act cat ttagta gga att aaa aat 204 Leu Lys Val Ala Glu Lys Thr Glu Thr His Leu ValGly Ile Lys Asn 35 40 45 aaa gat aat gaa gtc att gct gct tgt atg cta actgct gta ccc gtt 252 Lys Asp Asn Glu Val Ile Ala Ala Cys Met Leu Thr AlaVal Pro Val 50 55 60 atg aaa att ttt aaa tat ttt tat tca aat cgt ggt ccagtc att gat 300 Met Lys Ile Phe Lys Tyr Phe Tyr Ser Asn Arg Gly Pro ValIle Asp 65 70 75 tat gaa aac aaa gaa ctc gtt cac ttt ttc ttt aac gaa ttaagt aaa 348 Tyr Glu Asn Lys Glu Leu Val His Phe Phe Phe Asn Glu Leu SerLys 80 85 90 95 tat tta aaa caa caa cat tgt tta tat gta cgt ata gac ccttat ttg 396 Tyr Leu Lys Gln Gln His Cys Leu Tyr Val Arg Ile Asp Pro TyrLeu 100 105 110 cct tat caa tat cgt aat cat gat ggt gat att aca gga aatgct ggg 444 Pro Tyr Gln Tyr Arg Asn His Asp Gly Asp Ile Thr Gly Asn AlaGly 115 120 125 aat gat tgg ttc ttc gat aaa atg aaa caa tta gga tat caacac gaa 492 Asn Asp Trp Phe Phe Asp Lys Met Lys Gln Leu Gly Tyr Gln HisGlu 130 135 140 ggg ttt aca aca gga ttt gat cca ata tta caa att cgg ttccat tca 540 Gly Phe Thr Thr Gly Phe Asp Pro Ile Leu Gln Ile Arg Phe HisSer 145 150 155 gtt tta aat tta aag gat aaa act gct aaa gat gta tta aatgga atg 588 Val Leu Asn Leu Lys Asp Lys Thr Ala Lys Asp Val Leu Asn GlyMet 160 165 170 175 gat agt tta cga aaa aga aat act aaa aaa gtc caa aaaaat ggt gtt 636 Asp Ser Leu Arg Lys Arg Asn Thr Lys Lys Val Gln Lys AsnGly Val 180 185 190 aaa gta aga ttt ctt act aaa gaa gaa tta cct att ttcaga tca ttt 684 Lys Val Arg Phe Leu Thr Lys Glu Glu Leu Pro Ile Phe ArgSer Phe 195 200 205 atg gaa gat aca tca gag act aaa gaa ttt tct gat agagag gat agt 732 Met Glu Asp Thr Ser Glu Thr Lys Glu Phe Ser Asp Arg GluAsp Ser 210 215 220 ttt tac tat aat cga ttt gat cat ttt aaa gat aga gtatta gta cct 780 Phe Tyr Tyr Asn Arg Phe Asp His Phe Lys Asp Arg Val LeuVal Pro 225 230 235 ctc gca tat ata aaa ttt gat gaa tat ctt gaa gaa cttcat gca gaa 828 Leu Ala Tyr Ile Lys Phe Asp Glu Tyr Leu Glu Glu Leu HisAla Glu 240 245 250 255 cgt cag aca tta aat aaa gac tta aac aaa gct ctaaaa gat att gaa 876 Arg Gln Thr Leu Asn Lys Asp Leu Asn Lys Ala Leu LysAsp Ile Glu 260 265 270 aaa cga cca gat aac aaa aaa gca caa aat aaa aaaata aat tta gaa 924 Lys Arg Pro Asp Asn Lys Lys Ala Gln Asn Lys Lys IleAsn Leu Glu 275 280 285 cag caa tta aaa gca aat gag caa aaa att gat gaagca aca caa ctt 972 Gln Gln Leu Lys Ala Asn Glu Gln Lys Ile Asp Glu AlaThr Gln Leu 290 295 300 caa tta gaa cat ggt aac gaa tta cca ata tct gctgga ttc ttc ttt 1020 Gln Leu Glu His Gly Asn Glu Leu Pro Ile Ser Ala GlyPhe Phe Phe 305 310 315 att aat cca ttt gaa gtt gta tat tat gca ggt ggaacg tca aat aaa 1068 Ile Asn Pro Phe Glu Val Val Tyr Tyr Ala Gly Gly ThrSer Asn Lys 320 325 330 335 tat aga cac ttc gct gga agt tat gca gtt caatgg act atg att aat 1116 Tyr Arg His Phe Ala Gly Ser Tyr Ala Val Gln TrpThr Met Ile Asn 340 345 350 tat gca att gat cat ggc att gac cgt tat aatttt tat ggg att agt 1164 Tyr Ala Ile Asp His Gly Ile Asp Arg Tyr Asn PheTyr Gly Ile Ser 355 360 365 ggt cat ttt aca gat gat gct gaa gat gca ggtgtt gta aaa ttt aaa 1212 Gly His Phe Thr Asp Asp Ala Glu Asp Ala Gly ValVal Lys Phe Lys 370 375 380 aaa gga ttt aat gca gat gta att gaa tat gttggt gat ttc gtt aaa 1260 Lys Gly Phe Asn Ala Asp Val Ile Glu Tyr Val GlyAsp Phe Val Lys 385 390 395 cct ata aat aaa cca atg tat tca cta tat acaaca ctt aaa aaa att 1308 Pro Ile Asn Lys Pro Met Tyr Ser Leu Tyr Thr ThrLeu Lys Lys Ile 400 405 410 415 aaa aag aga ttgaattaag aggggaatag tgagaa1343 Lys Lys Arg 53 418 PRT Staphylococcus hominis femA 53 Met Lys PheThr Asn Leu Thr Ala Thr Glu Phe Gly Asp Phe Thr Glu 1 5 10 15 Lys MetPro Tyr Ser His Phe Thr Gln Met Thr Glu Asn Tyr Glu Leu 20 25 30 Lys ValAla Glu Lys Thr Glu Thr His Leu Val Gly Ile Lys Asn Lys 35 40 45 Asp AsnGlu Val Ile Ala Ala Cys Met Leu Thr Ala Val Pro Val Met 50 55 60 Lys IlePhe Lys Tyr Phe Tyr Ser Asn Arg Gly Pro Val Ile Asp Tyr 65 70 75 80 GluAsn Lys Glu Leu Val His Phe Phe Phe Asn Glu Leu Ser Lys Tyr 85 90 95 LeuLys Gln Gln His Cys Leu Tyr Val Arg Ile Asp Pro Tyr Leu Pro 100 105 110Tyr Gln Tyr Arg Asn His Asp Gly Asp Ile Thr Gly Asn Ala Gly Asn 115 120125 Asp Trp Phe Phe Asp Lys Met Lys Gln Leu Gly Tyr Gln His Glu Gly 130135 140 Phe Thr Thr Gly Phe Asp Pro Ile Leu Gln Ile Arg Phe His Ser Val145 150 155 160 Leu Asn Leu Lys Asp Lys Thr Ala Lys Asp Val Leu Asn GlyMet Asp 165 170 175 Ser Leu Arg Lys Arg Asn Thr Lys Lys Val Gln Lys AsnGly Val Lys 180 185 190 Val Arg Phe Leu Thr Lys Glu Glu Leu Pro Ile PheArg Ser Phe Met 195 200 205 Glu Asp Thr Ser Glu Thr Lys Glu Phe Ser AspArg Glu Asp Ser Phe 210 215 220 Tyr Tyr Asn Arg Phe Asp His Phe Lys AspArg Val Leu Val Pro Leu 225 230 235 240 Ala Tyr Ile Lys Phe Asp Glu TyrLeu Glu Glu Leu His Ala Glu Arg 245 250 255 Gln Thr Leu Asn Lys Asp LeuAsn Lys Ala Leu Lys Asp Ile Glu Lys 260 265 270 Arg Pro Asp Asn Lys LysAla Gln Asn Lys Lys Ile Asn Leu Glu Gln 275 280 285 Gln Leu Lys Ala AsnGlu Gln Lys Ile Asp Glu Ala Thr Gln Leu Gln 290 295 300 Leu Glu His GlyAsn Glu Leu Pro Ile Ser Ala Gly Phe Phe Phe Ile 305 310 315 320 Asn ProPhe Glu Val Val Tyr Tyr Ala Gly Gly Thr Ser Asn Lys Tyr 325 330 335 ArgHis Phe Ala Gly Ser Tyr Ala Val Gln Trp Thr Met Ile Asn Tyr 340 345 350Ala Ile Asp His Gly Ile Asp Arg Tyr Asn Phe Tyr Gly Ile Ser Gly 355 360365 His Phe Thr Asp Asp Ala Glu Asp Ala Gly Val Val Lys Phe Lys Lys 370375 380 Gly Phe Asn Ala Asp Val Ile Glu Tyr Val Gly Asp Phe Val Lys Pro385 390 395 400 Ile Asn Lys Pro Met Tyr Ser Leu Tyr Thr Thr Leu Lys LysIle Lys 405 410 415 Lys Arg 54 2742 DNA Staphylococcus saprophyticusfemA CDS (64)...(1326) 54 acttgtttag attagaatta aactcgaaaa tagaactatagataaatagg agtatataaa 60 aaa atg aaa ttt acg aat tta act gca aaa gag ttcggt gca ttt acg 108 Met Lys Phe Thr Asn Leu Thr Ala Lys Glu Phe Gly AlaPhe Thr 1 5 10 15 gat aaa atg ccg aat agt cat ttt acg caa atg gtt ggaaat tat gaa 156 Asp Lys Met Pro Asn Ser His Phe Thr Gln Met Val Gly AsnTyr Glu 20 25 30 ttg aaa att gca gaa agt aca gaa aca cac cta gta ggt attaag aat 204 Leu Lys Ile Ala Glu Ser Thr Glu Thr His Leu Val Gly Ile LysAsn 35 40 45 aat gat aat gaa gta att gca gca tgt tta ctt aca gct gtt cctgtt 252 Asn Asp Asn Glu Val Ile Ala Ala Cys Leu Leu Thr Ala Val Pro Val50 55 60 atg aaa ttc ttc aag tat ttt tat tcc aat aga ggt cca gtc ata gat300 Met Lys Phe Phe Lys Tyr Phe Tyr Ser Asn Arg Gly Pro Val Ile Asp 6570 75 ttt gaa aat aaa gaa ctc gta cat tac ttc ttt aac gaa tta gca aaa348 Phe Glu Asn Lys Glu Leu Val His Tyr Phe Phe Asn Glu Leu Ala Lys 8085 90 95 tat gta aaa aaa cat aat gcc tta tat tta cga gta gat cct tat ctt396 Tyr Val Lys Lys His Asn Ala Leu Tyr Leu Arg Val Asp Pro Tyr Leu 100105 110 gct tat caa tat cgt aat cat gat ggt gaa gta tta gca aat gcg ggt444 Ala Tyr Gln Tyr Arg Asn His Asp Gly Glu Val Leu Ala Asn Ala Gly 115120 125 cac gat tgg att ttt gat aaa atg aaa caa ctc ggt tat aag cat gaa492 His Asp Trp Ile Phe Asp Lys Met Lys Gln Leu Gly Tyr Lys His Glu 130135 140 ggt ttt tta act ggc ttt gac cca ata ctt caa ata aga ttc cat tct540 Gly Phe Leu Thr Gly Phe Asp Pro Ile Leu Gln Ile Arg Phe His Ser 145150 155 gtt tta gat tta gct gga aaa act gct aaa gac gta ctt aat ggt atg588 Val Leu Asp Leu Ala Gly Lys Thr Ala Lys Asp Val Leu Asn Gly Met 160165 170 175 gat agt tta cgt aaa cga aat act aaa aaa gta cag aaa aat ggtgtg 636 Asp Ser Leu Arg Lys Arg Asn Thr Lys Lys Val Gln Lys Asn Gly Val180 185 190 aaa gta aga ttt tta ggt gaa gat gag ttg cca ata ttc cgc tcattc 684 Lys Val Arg Phe Leu Gly Glu Asp Glu Leu Pro Ile Phe Arg Ser Phe195 200 205 atg gaa gat act tct gaa aca aag gat ttt gac gat aga gat gacgat 732 Met Glu Asp Thr Ser Glu Thr Lys Asp Phe Asp Asp Arg Asp Asp Asp210 215 220 ttt tat tat aat agg tta aga tat tat aaa gat cgt gtg ctt gtccca 780 Phe Tyr Tyr Asn Arg Leu Arg Tyr Tyr Lys Asp Arg Val Leu Val Pro225 230 235 tta gct tat atg gat ttt gat gaa tat ata aca gaa tta aag gctgaa 828 Leu Ala Tyr Met Asp Phe Asp Glu Tyr Ile Thr Glu Leu Lys Ala Glu240 245 250 255 cgc gaa gta tta agt aaa gat ata aat aaa gca gtt aag gatata gaa 876 Arg Glu Val Leu Ser Lys Asp Ile Asn Lys Ala Val Lys Asp IleGlu 260 265 270 aaa aga cca gaa aat aaa aaa gcg tat aat aaa aaa gaa aattta gaa 924 Lys Arg Pro Glu Asn Lys Lys Ala Tyr Asn Lys Lys Glu Asn LeuGlu 275 280 285 caa caa ctg att gca aac caa caa aaa ata gat gaa gcc actgcg tta 972 Gln Gln Leu Ile Ala Asn Gln Gln Lys Ile Asp Glu Ala Thr AlaLeu 290 295 300 caa gag aag cat ggt aac gaa tta ccg att tct gca gct tacttt att 1020 Gln Glu Lys His Gly Asn Glu Leu Pro Ile Ser Ala Ala Tyr PheIle 305 310 315 att aat cct tat gaa gtc gtt tac tat gca ggt ggt aca tctaat gaa 1068 Ile Asn Pro Tyr Glu Val Val Tyr Tyr Ala Gly Gly Thr Ser AsnGlu 320 325 330 335 ttt aga cat ttt gct ggt agt tat gca ata caa tgg aagatg att aat 1116 Phe Arg His Phe Ala Gly Ser Tyr Ala Ile Gln Trp Lys MetIle Asn 340 345 350 tat gct ata gat cat aat ata gat aga tat aat ttt tatggt att agt 1164 Tyr Ala Ile Asp His Asn Ile Asp Arg Tyr Asn Phe Tyr GlyIle Ser 355 360 365 ggt cat ttt act gaa gat gca gaa gat gca ggt gtt gttaaa ttt aaa 1212 Gly His Phe Thr Glu Asp Ala Glu Asp Ala Gly Val Val LysPhe Lys 370 375 380 aaa ggt ttt aat gca gat gta gta gaa tat gtt ggt gatttt att aaa 1260 Lys Gly Phe Asn Ala Asp Val Val Glu Tyr Val Gly Asp PheIle Lys 385 390 395 ccg att aat aag cca atg tac aaa att tat acg aca ttgaaa aaa att 1308 Pro Ile Asn Lys Pro Met Tyr Lys Ile Tyr Thr Thr Leu LysLys Ile 400 405 410 415 aag gat aaa aag aaa taa acataaatag aagggaactaagctagaatg 1356 Lys Asp Lys Lys Lys * 420 aaatttacag agttaacttgtttagattag aattaaactc gaaaatagaa ctatagataa 1416 ataggagtat ataaaaaaatgaaatttacg aatttaactg caaaagagtt cggtgcattt 1476 acggataaaa tgccgaatagtcattttacg caaatggttg gaaattatga attgaaaatt 1536 gcagaaagta cagaaacacacctagtaggt attaagaata atgataatga agtaattgca 1596 gcatgtttac ttacagctgttcctgttatg aaattcttca agtattttta ttccaataga 1656 ggtccagtca tagattttgaaaataaagaa ctcgtacatt acttctttaa cgaattagca 1716 aaatatgtaa aaaaacataatgccttatat ttacgagtag atccttatct tgcttatcaa 1776 tatcgtaatc atgatggtgaagtattagca aatgcgggtc acgattggat ttttgataaa 1836 atgaaacaac tcggttataagcatgaaggt tttttaactg gctttgaccc aatacttcaa 1896 ataagattcc attctgttttagatttagct ggaaaaactg ctaaagacgt acttaatggt 1956 atggatagtt tacgtaaacgaaatactaaa aaagtacaga aaaatggtgt gaaagtaaga 2016 tttttaggtg aagatgagttgccaatattc cgctcattca tggaagatac ttctgaaaca 2076 aaggattttg acgatagagatgacgatttt tattataata ggttaagata ttataaagat 2136 cgtgtgcttg tcccattagcttatatggat tttgatgaat atataacaga attaaaggct 2196 gaacgcgaag tattaagtaaagatataaat aaagcagtta aggatataga aaaaagacca 2256 gaaaataaaa aagcgtataataaaaaagaa aatttagaac aacaactgat tgcaaaccaa 2316 caaaaaatag atgaagccactgcgttacaa gagaagcatg gtaacgaatt accgatttct 2376 gcagcttact ttattattaatccttatgaa gtcgtttact atgcaggtgg tacatctaat 2436 gaatttagac attttgctggtagttatgca atacaatgga agatgattaa ttatgctata 2496 gatcataata tagatagatataatttttat ggtattagtg gtcattttac tgaagatgca 2556 gaagatgcag gtgttgttaaatttaaaaaa ggttttaatg cagatgtagt agaatatgtt 2616 ggtgatttta ttaaaccgattaataagcca atgtacaaaa tttatacgac attgaaaaaa 2676 attaaggata aaaagaaataaacataaata gaagggaact aagctagaat gaaatttaca 2736 gagtta 2742 55 420 PRTStaphylococcus saprophyticus femA 55 Met Lys Phe Thr Asn Leu Thr Ala LysGlu Phe Gly Ala Phe Thr Asp 1 5 10 15 Lys Met Pro Asn Ser His Phe ThrGln Met Val Gly Asn Tyr Glu Leu 20 25 30 Lys Ile Ala Glu Ser Thr Glu ThrHis Leu Val Gly Ile Lys Asn Asn 35 40 45 Asp Asn Glu Val Ile Ala Ala CysLeu Leu Thr Ala Val Pro Val Met 50 55 60 Lys Phe Phe Lys Tyr Phe Tyr SerAsn Arg Gly Pro Val Ile Asp Phe 65 70 75 80 Glu Asn Lys Glu Leu Val HisTyr Phe Phe Asn Glu Leu Ala Lys Tyr 85 90 95 Val Lys Lys His Asn Ala LeuTyr Leu Arg Val Asp Pro Tyr Leu Ala 100 105 110 Tyr Gln Tyr Arg Asn HisAsp Gly Glu Val Leu Ala Asn Ala Gly His 115 120 125 Asp Trp Ile Phe AspLys Met Lys Gln Leu Gly Tyr Lys His Glu Gly 130 135 140 Phe Leu Thr GlyPhe Asp Pro Ile Leu Gln Ile Arg Phe His Ser Val 145 150 155 160 Leu AspLeu Ala Gly Lys Thr Ala Lys Asp Val Leu Asn Gly Met Asp 165 170 175 SerLeu Arg Lys Arg Asn Thr Lys Lys Val Gln Lys Asn Gly Val Lys 180 185 190Val Arg Phe Leu Gly Glu Asp Glu Leu Pro Ile Phe Arg Ser Phe Met 195 200205 Glu Asp Thr Ser Glu Thr Lys Asp Phe Asp Asp Arg Asp Asp Asp Phe 210215 220 Tyr Tyr Asn Arg Leu Arg Tyr Tyr Lys Asp Arg Val Leu Val Pro Leu225 230 235 240 Ala Tyr Met Asp Phe Asp Glu Tyr Ile Thr Glu Leu Lys AlaGlu Arg 245 250 255 Glu Val Leu Ser Lys Asp Ile Asn Lys Ala Val Lys AspIle Glu Lys 260 265 270 Arg Pro Glu Asn Lys Lys Ala Tyr Asn Lys Lys GluAsn Leu Glu Gln 275 280 285 Gln Leu Ile Ala Asn Gln Gln Lys Ile Asp GluAla Thr Ala Leu Gln 290 295 300 Glu Lys His Gly Asn Glu Leu Pro Ile SerAla Ala Tyr Phe Ile Ile 305 310 315 320 Asn Pro Tyr Glu Val Val Tyr TyrAla Gly Gly Thr Ser Asn Glu Phe 325 330 335 Arg His Phe Ala Gly Ser TyrAla Ile Gln Trp Lys Met Ile Asn Tyr 340 345 350 Ala Ile Asp His Asn IleAsp Arg Tyr Asn Phe Tyr Gly Ile Ser Gly 355 360 365 His Phe Thr Glu AspAla Glu Asp Ala Gly Val Val Lys Phe Lys Lys 370 375 380 Gly Phe Asn AlaAsp Val Val Glu Tyr Val Gly Asp Phe Ile Lys Pro 385 390 395 400 Ile AsnLys Pro Met Tyr Lys Ile Tyr Thr Thr Leu Lys Lys Ile Lys 405 410 415 AspLys Lys Lys 420 56 18 DNA Artificial Sequence Primer 56 ttcmaatcgcggtccagt 18 57 23 DNA Artificial Sequence Primer 57 caagaacatggcaacgaatt acc 23 58 23 DNA Artificial Sequence Primer 58 tgggtaattcgttgccatgt tct 23 59 21 DNA Artificial Sequence Primer 59 ccaagcatcttcagcatctt c 21 60 29 DNA Artificial Sequence Primer 60 ttctttaactgttaactctg taaatttca 29 61 26 DNA Artificial Sequence Primer 61acatatttac ttaattcgtt aaagaa 26 62 27 DNA Artificial Sequence Primer 62cagaaaaatg gtgttaaagt aagattt 27 63 27 DNA Artificial Sequence Primer 63aagaaatctt actttcacac cattttt 27 64 18 DNA Artificial Sequence Primer 64aactcgaaaa tagaacta 18 65 20 DNA Artificial Sequence Universal primer 65gccatacagt catttcacgc 20 66 18 DNA Artificial Sequence femA primer foridentification of S. aureus 66 cagcagatga catcatta 18 67 18 DNAArtificial Sequence femA primer for identification of S. haemolyticus 67aaccgaaacg aaagaatt 18 68 21 DNA Artificial Sequence femA primer foridentification of S. hominis 68 caacacaact tcaattagaa c 21 69 27 DNAArtificial Sequence primer/probe for identification of S. aureus sbs.anaerobius 69 atttaaaata tcacgctctt cgtttag 27 70 27 DNA ArtificialSequence primer/probe for identification of S. epidermidis 70 attaagcacatttctttcat tatttag 27 71 27 DNA Artificial Sequence primer/probe foridentification of S. haemolyticus 71 atttaaagtt tcacgttcat tttgtaa 27 7227 DNA Artificial Sequence primer/probe for identification of S. hominis72 atttaatgtc tgacgttctg catgaag 27 73 27 DNA Artificial Sequenceprimer/probe for identification of S. saprophyticus 73 acttaatacttcgcgttcag cctttaa 27 74 27 DNA Artificial Sequence primer/probe foridentification of S. capitis 74 attaagaaca tctctttcat tattaag 27 75 27DNA Artificial Sequence primer/probe for identification of S. cohniisbs.urealyticum 75 acttaacact tcacgctctg acttgag 27 76 27 DNA ArtificialSequence primer/probe for identification of S. gallinarum 76 acttaaaacttcacgttcag cagtaag 27 77 27 DNA Artificial Sequence primer/probe foridentification of S. intermedius 77 gtggaaatct tgctcttcag atttcag 27 7827 DNA Artificial Sequence primer/probe for identification of S.lugdunensis 78 ttctaaagtt tgtcgttcat tcgttag 27 79 27 DNA ArtificialSequence primer/probe for identification of S. schleiferi 79 tttaaagtcttgcgcttcag tgttgag 27 80 27 DNA Artificial Sequence primer/probe foridentification of S. sciuri 80 gttgtattgt tcatgttctt tttctaa 27 81 27DNA Artificial Sequence primer/probe for identification of S. simulans81 ttctaaattc ttttgttcag cgttcaa 27 82 27 DNA Artificial Sequenceprimer/probe for identification of S. warneri 82 agttaaggtt tctttttcattattgag 27 83 27 DNA Artificial Sequence primer/probe for identificationof S. xylosus 83 gcttaacacc tcacgttgag cttgcaa 27

What is claimed is:
 1. An isolated and purified nucleotide sequencecorresponding to SEQ ID NO:1, homologues or parts thereof.
 2. Anisolated and purified nucleotide sequence according to claim 1, whereinsaid homologues comprise SEQ ID NOs: 40, 42, 44, 46, 48, 50, 52 and 54.3. An isolated and purified nucleotide sequence according to claim 2,wherein said homologues have more than 85% homology to SEQ ID NOs: 40,42, 44, 46, 48, 50, 52 and
 54. 4. An isolated and purified nucleotidesequence according to claim 2, wherein said homologues have more than90% homology to SEQ ID NOs: 40, 42, 44, 46, 48, 50, 52 and
 54. 5. Anisolated and purified nucleotide sequence according to claim 1, whereinsaid parts of SEQ ID NO:1 are nucleotide sequences having between 25 and350 bp, which have more than 60% homology to SEQ ID NO:1.
 6. An isolatedand purified nucleotide sequence according to claim 1, wherein saidparts of SEQ ID NO:1 are nucleotide sequences having between 25 and 250bp, which have more than 60% homology to SEQ ID NO:1.
 7. An isolated andpurified nucleotide sequence according to claim 1, wherein said parts ofSEQ ID NO:1 are nucleotide sequences having between 25 and 45 bp, whichhave more than 60% homology to SEQ ID NO:1.
 8. An isolated and purifiednucleotide sequence according to claim 1, wherein said parts of SEQ IDNO:1 comprise SEQ ID NOS: 2-28, 56-64.
 9. An isolated and purifiednucleotide sequence according to claim 1, wherein said parts of SEQ IDNO:1 comprise SEQ ID NOS: 65-83.
 10. Use of an isolated and purifiednucleotide sequence corresponding to SEQ ID NO:1, homologues or partsthereof for the identification of at least two Staphylococci species.11. Diagnostic device for the identification of at least twoStaphylococci species comprising at least one oligonucleotide which hasmore than 60% homology to SEQ ID NO:1 and at least one isolated orpurified oligonucleotides which has less than 50% homology to SEQ IDNO:1.